Furanone derivatives

ABSTRACT

The invention relates to a method of treating with furanone derivatives and the pharmaceutically acceptable salts thereof, one or more conditions of the skin, in particular regulating skin condition, regulating signs of skin aging, treating a number of conditions such as contact dermatitis, skin irritation, rosacea, acne, psoriasis, and preventing and protecting skin tissue against age-related damage or damage resulting from insults such as harmful (UV) radiation or environmental pollution, stress and fatigue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of non-provisionalapplication Ser. No. 10/354,474 filed on Jan. 30, 2003 now U.S. Pat. No.6,667,330 claiming priority under 35 U.S.C. 119(e) to U.S. ProvisionalApplication Ser. No. 60/353,939, filed Jan. 31, 2002, incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to furanone derivatives, particularly toderivatives having cytoprotective activity, especially certain3-hydroxy-furan-2-one derivatives. The invention is also directed toformulations and methods for treating stroke, myocardial infarction andchronic heart failure, as well as other oxidative stress-relatedconditions that are typically responsive to cellular enzyme modulation.The invention is also directed to formulations and methods for treatingneuroinflammation, cognitive disorders and neurodegenerative diseasessuch a Alzheimer's disease and senile dementia. This invention is alsodirected to methods for treating dermatological conditions.

BACKGROUND INFORMATION

The present invention deals with certain novel furanone derivatives,which are formed under proper conditions from a series of pyruvatederivatives described in our prior applications, U.S. Ser. Nos.10/138,937 and 10/138,032.

Furanones are compounds having the following general structure.

Furanone-derived compositions have been known in the art to have variousutilities. For example, U.S. Pat. No. 6,296,889 describes the use ofcertain furanone compounds in conjunction with 1-nonen-3-one to providedairy and coffee aroma flavor enhancement. Specific furanones (forexample,3,-(3,4-difluorophenyl)-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone,3-phenyl-4-(4-(methylsulfonyl)phenyl)-2-(5H)-furanone and5,5-dimethyl-4-(4-(methylsulfonyl)phenyl)-3-(3-fluorophenyl)-5H-furan-2-one)have been shown to be cyclooxygenase-2 (COX-2) inhibitors useful intreating certain inflammatory conditions (U.S. Pat. Nos. 5,474,995,6,239,173). The diversity of furanone derivative utilities is furtherillustrated by the discovery of certain halogenated furanones isolatedfrom the Australian red seaweed Delisea nulcha as marine anti-foulingagents (U.S. Pat. No. 6,060,046) capable of preventing growth of variousseaweeds, invertebrates and bacteria on marine structures. The furanonederivative4-hydroxy-3-methanesulfonyl-2-methanesulfonylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester (CAS Registry No. 299923-61-8), is available forscreening from the compound library of InterBioScreen Ltd. (Moscow,Russia-www.ibscreen.com), among other sources.

The synthesis of the compound4-hydroxy-3-isobutyl-2-(3-methyl-butyryl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid methyl ester has been described in Trogolo, C. et al Annali diChimica 62(10), 674-92, (1972) and the synthesis of4-hydroxy-5-oxo-2,3-dipentyl-2,5-dihydro-furan-2-carboxylic acid ethylester has been described in Hoffman, R. V. et al. Journal of OrganicChemistry, 62(8)2458-2465, (1997). The synthesis of certain furanonoesderivatives from hydroxy alkanoates is described in Stach, H., HelveticaChimica Acta (1987), 70(2), 369-74.

Cerebral ischemia or “stroke” refers to the severe diminution orcessation of blood flow to all or part of the brain. Cerebral ischemiacan occur as result of a number of causes or insults, including, but notlimited to cerebrovascular occlusion, thromboembolytic insult, cardiacfailure and hemorrhagic accident. It is now known that pharmacologicintervention, if provided within a reasonable interval of the initialinsult, can significantly reduce cerebral tissue death followingcerebral ischemia.

Alzheimer's Disease (“AD”) is a progressive disease of the human centralnervous system. It is manifested by dementia in the elderly, bydisorientation, loss of memory, difficulty with language, calculation,or visual-spatial skills, and by psychiatric manifestations. It isassociated with degenerating neurons in several regions of the brain.Alzheimer's Disease is reviewed by Price, D. L. et al. (Clin.Neuropharm. 14:S9-S14 (1991)); Pollwein, P. et al. (Nucl. Acids Res.20:63-68 (1992)); Regland, B. et al. (Med. Hypoth. 38:11-19 (1992)) andJohnson, S. A. (In: Review of Biological Research in Aging, Vol. 4.,Rothstein, M. (Ed.), Wiley-Liss, NY, 163-170 (1990)).

The present invention addresses the desire to provide the therapies forconditions characterized by oxidative stress and/or inflammation, andparticularly, for providing neuroprotection in the event of cerebralischemia; especially desired are agents that are effective even if firstadministered after a significant period of time (e.g., about 5 or morehours) following an ischemic insult. The present invention alsoaddresses the desire to provide new therapies for conditionscharacterized by neuroinflammation, cognitive disorders, and/orneurodegenerative conditions such as Alzheimer's and senile dementia, aswell as dermatological disorders or diseases.

SUMMARY OF THE INVENTION

The present invention is concerned with novel furanone derivatives thatare particularly active in restoring or preserving metabolic integrityin oxidatively competent cells that have been subjected to oxygendeprivation. Such furanone derivatives are useful in the manufacture ofpharmaceutical compositions for treating a number of conditionscharacterized by oxidative stress, and particularly, in providingneuroprotection in the event of cerebral ischemia, even whenadministered a significant time interval after an ischemic insult. Inparticular, the compositions of the present invention are useful in thetreatment of stroke, as demonstrated by providing neuroprotection in astandard experimental model of focal cerebral ischemia. They are alsouseful in the treatment of neuroinflammation, cognitive disorders andneurodegenerative diseases such as neuropathy in cerebrovasculardiseases, brain trauma, cerebral palsy, epilepsy, amyotrophic lateralsclerosis (ALS), Huntington's disease, mental diseases (e.g. psychosis,schizophrenia, depression), Parkinson's disease, Friedreich's disease,Down's syndrome, Creutzfeldt-Jakob syndrome, Alzheimer's disease, andsenile dementia.

They are also useful in the treatment of myocardial ischemia (myocardialinfarction), as well as other indications characterized by oxidativestress and/or inflammation, including, but not limited to, diabetes,renal disease, pre-menstrual syndrome, asthma, cardiopulmonaryinflammatory disorders, chronic heart failure, rheumatoid arthritis,muscle fatigue, intermittent claudication and for the preservation ofallograft tissue for transplantation.

The compounds of the present invention are useful for the treatment ofdermatological conditions, particularly the compounds, formulation andmethods of the present invention are useful in regulating skincondition, regulating signs of skin aging and in treating a number ofconditions, including but not limited to contact dermatitis, skinirritation, rosacea, acne, psoriasis, and in the prevention andprotection of skin tissue against age-related damage or damage resultingfrom insults such as harmful (UV) radiation or environmental pollution,stress and fatigue.

The present invention concerns the compounds represented by the formula:

wherein:

-   -   R¹ is: —C(O)OR′; —C(O)NR′R″; —CH₂OR′″; cyano; optionally        substituted heterocyclyl; optionally substituted        heterocyclyl-alkyl; optionally substituted heteroaryl, or        optionally substituted heteroaralkyl;    -   R² is: optionally substituted alkyl; optionally substituted        cycloalkyl; optionally substituted aryl; optionally substituted        aralkyl; optionally substituted heterocyclyl, optionally        substituted heteroaryl; optionally substituted heteroaralkyl; an        optionally substituted nucleoside; and optionally substituted        amino acid; or an optionally substituted di-, tri- or        tetra-peptide;    -   R³ is: optionally substituted alkyl; optionally substituted        cycloalkyl; optionally substituted aryl; optionally substituted        aralkyl; optionally substituted heterocyclyl, optionally        substituted heteroaryl; optionally substituted heteroaralkyl; an        optionally substituted nucleoside; an optionally substituted        amino acid; or an optionally substituted di-, tri- or        tetra-peptide;    -   R⁴ is: hydrogen; alkyl, alkylcarbonyl; (poly)alkoxyalkylene; or        dialkoxyphosphoryloxy (or other moieties readily hydrolyzable to        give an OH moiety);    -   X is: lower alkylene; —N(R′)—; —S—; —S(O)—; —S(O)₂—, or X taken        together with R² is —P(O)(OR′)₂;    -   Y is: —N(R′)—; —S—; —S(O)—; —S(O)₂—, or Y taken together with R³        is —P(O)(OR′)₂;    -   or X—R² taken together with Y—R³ form an optionally substituted        aliphatic or aromatic ring,    -   R′ is: hydrogen; alkenyl; optionally substituted alkyl;        optionally substituted cycloalkyl; phosphoryl; or optionally        substituted aryl;    -   R″ is: hydrogen, alkenyl, optionally substituted alkyl, or        optionally substituted aryl;    -   or R′ and R″ together with the atom to which they are attached        form a 5- to 7-membered aromatic, saturated or unsaturated ring,        optionally incorporating one or more additional heteroatom        chosen from N, O, or S, and optionally substituted with one or        more substituents selected from the group consisting of        optionally substituted lower alkyl, halo, cyano, alkylthio,        lower alkoxy, carboxy, benzyl, and oxo; and    -   R′″ is: hydrogen; alkenyl; optionally substituted alkyl;        optionally substituted cycloalkyl; acyl; phosphoryl; or        optionally substituted aryl;        including single tautomers, single stereoisomers and mixtures of        tautomers and/or stereoisomers, and the pharmaceutically        acceptable salts thereof.

In one embodiment R⁴ is hydrogen, (C₁ to C₈)alkyl, or (C₁ toC₈)alkylcarbonyl.

In another embodiment where R² and/or R³ is a natural or substitutedamino acid or peptide, R² and/or R³ is selected from the group: Ala,Asn, Asp, Cys, Gin, Glu, Gly, Lys, Met, Ser and Thr, especially Ala,Asp, Cys, Glu and Gly. Further preferred are those compounds where R²and/or R³ is a natural or substituted di- or tri-peptide, especiallynatural peptides.

In yet another embodiment, R² and/or R³ is/are an optionally substitutedheteroaryl or heteroaralkyl group, especially a nitrogen-containingoptionally substituted heteroaryl, and particularly benzooxazole,benzoselenazol, and benzothiazole, or an optionally substitutedheteroaralkyl group, particularly an optionally substitutedfuranyl-loweralkyl group.

In yet another embodiment, R² and/or R³ is/are an optionally substitutedalkyl or optionally substituted cycloalkyl.

In another embodiment, R² and/or R³ is/are an optionally substitutedaryl or optionally substituted aralkyl, preferably optionallysubstituted phenyl or benzyl.

Further preferred in each of the foregoing embodiments, R¹ is —C(O)OR′,—CH₂OR′″ or —C(O)NR′R″; and R′, R″, and R′″ are hydrogen or lower alkyl(C₁ to C₈), and especially R′ is —C(O)OR′, and R′ is hydrogen or (C₁ toC₈)alkyl.

Presently preferred for the pharmaceutically acceptable salts of theinvention are the TEA, TFA, HCl, HBr, MsOH, TsOH, AcOH, and Na salts ofthe furanone compounds of the present invention.

Further preferred in each of the foregoing embodiments are thosecompounds where X and Y are the same particularly —S—, —S(O)— or—S(O)₂—, preferably —S—; and especially those where —X—R² and —Y—R³ arethe same.

Another embodiment of the invention concerns compounds according toFormula I where X—R² and/or Y—R³ is/are represented by the formula:

where:

-   -   R^(2.1) is: hydrogen, optionally substituted alkyl, optionally        substituted aryl, —C(O)—O—R²′, —S—, or —CH₂—S—;    -   R^(2.2) is: hydrogen, optionally substituted alkyl, optionally        substituted aryl, optionally substituted aralkyl, or optionally        substituted acyl (particularly including aliphatic, aromatic and        cyclic    -   R^(2.3) is: hydrogen, optionally substituted lower alkyl,        optionally substituted aralkyl, optionally substituted        heteroaralkyl, or —CH₂—S— (selected independently, in each        occurrence of R^(2.3));    -   R^(2.4) is: hydrogen, optionally substituted lower alkyl,        optionally substituted aralkyl, optionally substituted        heteroaralkyl, or —CH₂—S—;    -   R^(2.5) is: hydrogen, optionally substituted alkyl, or        optionally substituted aryl;    -   R²′ is: hydrogen, optionally substituted alkyl, or optionally        substituted aryl (selected independently, in each occurrence of        R²′);    -   k is: 0, 1 or 2;    -   m is: 0, 1 or 2; and    -   n is: 0, 1, 2 or 3,        at least one of R^(2.1), R^(2.3) and R^(2.4) being —CH₂—S—.

Of the compounds where X—R² and/or Y—R³ are represented by Formula II,preferred are those compounds the substituents of which are selectedfrom the following groups:

-   -   R^(2.1) is —C(O)—O—R²′ where R²′ is hydrogen or lower alkyl,        especially ethyl;    -   R^(2.2) is hydrogen;    -   R^(2.3) is —CH₂—S—;    -   R^(2.4) is hydrogen, optionally substituted lower alkyl,        optionally substituted aralkyl, optionally substituted        heteroaralkyl;    -   R^(2.5) is hydrogen or lower alkyl, especially hydrogen; and/or    -   k, m and n are respectively: 0,2,1; 1,0,1; or 2,0,1.

Another aspect of this invention concerns the compounds represented bythe formula:

wherein:

-   -   R⁵ is: —C(O)OR^(a); —C(O)NR^(a)R^(b); —CH₂OR^(d); —C(O)R^(c);        cyano; optionally substituted heterocyclyl, or optionally        substituted heteroaryl;    -   R⁶ is hydrogen; —C(O)OR^(a); —C(O)NR^(a)R^(b); —CH₂OR^(d);        —C(O)R^(c); cyano; optionally substituted alkyl; optionally        substituted heterocyclyl; optionally substituted aryl; or        optionally substituted heteroaryl;    -   or R⁵ and R⁶ with the atom to which they are attached form an        optionally substituted ring;    -   R⁷ is: optionally substituted alkyl; optionally substituted        cycloalkyl; optionally substituted aryl; optionally substituted        aralkyl; optionally substituted heterocyclyl, optionally        substituted heteroaryl; optionally substituted heteroaralkyl; an        optionally substituted nucleoside; an optionally substituted        amino acid; or an optionally substituted di-, tri- or        tetra-peptide; with the proviso that when R⁶ is alkyl, then R⁷        is optionally substituted heterocyclyl, optionally substituted        heteroaryl, or optionally substituted heteroaralkyl.    -   or R⁵ and R⁷ with the atoms to which they are attached form an        optionally substituted heterocyclic ring;    -   R⁸ is: hydrogen; alkyl, alkylcarbonyl; (poly)alkoxyalkylene; or        dialkoxyphosphoryloxy;    -   Y′ is: —N(R^(a))—; —S—; —S(O)—; or —S(O)₂—;    -   R^(a) is: hydrogen; alkenyl; optionally substituted alkyl;        optionally substituted cycloalkyl; or optionally substituted        aryl;    -   R^(b) is: hydrogen; alkenyl; optionally substituted alkyl; alkyl        ether; or optionally substituted aryl;    -   or R^(a) and R^(b) together with the atom to which they are        attached form a 5- to 7-membered aromatic, saturated or        unsaturated ring, optionally incorporating one more additional        heteroatom chosen from N, O, or S, and optionally substituted        with one or more substituents selected from the group consisting        of optionally substituted lower alkyl, halo cyano alkylthio,        lower alkoxy, carboxy, benzyl, and oxo;    -   R^(c) is optionally substituted alkyl or optionally substituted        aryl; and    -   R^(d) is hydrogen; alkenyl; optionally substituted alkyl; acyl,        optionally substituted cycloalkyl; or optionally substituted        aryl;        including single tautomers, single stereoisomers and mixtures of        tautomers and/or stereoisomers, and the pharmaceutically        acceptable salts thereof.

In another embodiment, R⁷ is an optionally substituted heteroaryl group,especially a nitrogen-containing optionally substituted heteroaryl, andparticularly selected from the group: imidazole; pyrazole; triazole;thiadiazole; oxadiazole; benzoimidazole; benzooxazole; benzoselenazoleand benzothiazole.

In another embodiment R⁵ is —C(O)OR^(a) or —C(O)R^(c); R^(a) ishydrogen, (C₁-C₈)alkyl, or (C₃-C₈)cycloalkyl; and R^(c) is lower alkylor aryl.

In another embodiment R⁵ is heteroaryl and R⁶ is hydrogen.

In another preferred embodiment R⁶ is hydrogen or —C(O)OR^(a); and R^(a)is hydrogen or lower alkyl (C₁ to C₈).

In another preferred embodiment R⁵ and R⁶ form a ring, particularly apyrimidine-2,4,6-trione ring or a cyclohexanone ring.

In another preferred embodiment R⁵ and R⁷ form a ring, particularly whenR⁷ is benzoimidazole, the ring is 3-methyl-thiomorpholin-3-ol and thecompound formed is1,4-dihydro-4-methyl-3a,4-dihydro-3-oxa-10-thia-4a,9-diaza-cyclopenta[b]fluoren-2-one.

In another preferred embodiment R⁸ is hydrogen.

Further preferred in each of the foregoing embodiments are thosecompounds where R⁵ is —C(O)OR^(a) or —C(O)R^(c), R⁶ is hydrogen, and Y′is —S—.

In another aspect the invention relates to compounds of Formula I orFormula III forming a complex with a metal, especially when said metalis selected form divalent copper, manganese, or zinc, particularlywherein said metals are selected from Cu²⁺Cl₂, Mn²⁺Cl₂, and Zn²⁺Cl₂.

In another aspect, the invention relates to a pharmaceutical compositioncontaining a therapeutically effective amount of a compound of Formula Ior Formula III, or a pharmaceutically acceptable salt thereof admixedwith at least one pharmaceutically acceptable excipient. Particularlypreferred are those pharmaceutical compositions wherein the compound ofFormula I or Formula III is selected from the preferred compounds.

In still another aspect, the invention relates to a method of treatingstroke and/or other oxidative stress-related conditions that areresponsive to cellular enzyme modulation such as cerebral ischemia,myocardial infarction and chronic heart failure (especiallystroke/cerebral ischemia) in a mammal by administering to a mammal inneed of such treatment a therapeutically effective amount of a compoundof Formula I or Formula III or a pharmaceutically acceptable saltthereof. Particularly preferred are those methods of treatment and usesin the manufacture of pharmaceutical compositions thereof, wherein thecompound of Formula I or Formula III is selected from the preferredcompounds, and especially from the compounds selected from:

-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid (2-hydroxy-ethyl)-amide;-   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   4-(2,2-Dimethyl-propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzooxazol-2-ylsulfanyl)-2-(benzooxazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-dihydro-furan-2-carboxylic    acid;-   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyridin-4-ylsulfanyl)-2-(pyridin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanyl)-2-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanymethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

In a preferred embodiment the invention relates to methods of treating acondition selected from stroke, cerebral ischemia, retinal ischemia,post-surgical cognitive dysfunctions, peripheral neuropathy/neuropathicpain, spinal cord injury, head injury and surgical trauma.

In another preferred embodiment the invention relates to methods oftreating a condition involving inflammatory or autoimmune components,especially diseases including but not limited to diabetes, renaldisease, premenstrual syndrome, asthma, rheumatoid arthritis,osteoarthritis, muscle fatigue, irritable bowel syndrome, inflammatorybowel disease, and intermittent claudication. Particularly preferred arethose methods of treatment and uses in the manufacture of pharmaceuticalcompositions therefore, wherein the compound is selected from thepreferred compounds, and especially from the compounds selected from;

-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-sulfonic    acid-1H-benzoimidazol-2-ylsulfanyl)-2-(5-sulfonic    acid-1H-benzoimidazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester, hydrochloride salt;-   4-Hydroxy-5-oxo-3-(pyridin-4-ylsulfanyl)-2-(pyridin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   5,8-Dichloro-3-hydroxy-2-oxo-2H-1-oxa-4,9-dithia-benzo[f]azulene-10a-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;

In still another aspect, the invention relates to a method of treatingneuroinflammation, cognitive disorders, and/or neurodegenerativedisorders in a mammal by administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of Formula Ior Formula III, or a pharmaceutically acceptable salt thereof.Particularly preferred are those methods of treatment and uses in themanufacture of pharmaceutical and/or cosmetic compositions thereof,wherein the compound of Formula I or Formula III is selected from thepreferred compounds, and especially from the compounds selected from:

-   3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(Benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-Dimethyl-propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methoxy-benzylsulfanyl)-2-(4-methoxy-benzylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methyl-pyrimidin-2-ylsulfanyl)-2-(4-methyl-pyrimidin-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

Another group of diseases characterized by oxidative stress fall withinthe group dermatologic conditions, including, but not limited toprevention and protecting skin tissue against age-related damage ordamage resulting from insults such as harmful ultraviolet (UV)radiation, stress and fatigue, and in the treatment of contactdermatitis, skin irritation, skin pigmentation, psoriasis, rosacea, oracne. In a method for regulating skin condition, regulating the signs ofskin aging or for treating contact dermatitis, skin irritation, acne,rosacea, psoriasis, age-related damage or damage resulting from harmful(UV) radiation or environmental pollution, stress or fatigue, theinvention entails topically administering to a mammal in need of suchtreatment a therapeutically effective amount of a compound of Formula I,Formula II, or Formula III or a pharmaceutically acceptable saltthereof. Particularly preferred are those methods of treatment and usesin the manufacture of pharmaceutical compositions therefore, wherein thecompound is selected from the preferred compounds, and especially fromthe compounds selected

-   R-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-oxy-pyridin-2-ylsulfanyl)-2-(1-oxy-pyridin-2-ylsulfanylmethyl)-2,5-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-5-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(Benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   Dimethylamino-acetic acid    3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethyl    ester;-   3-(2-Chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(5,6-Dichloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5,6-dichloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4.5]dec-3-ene-2,6,8,10-tetraone;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid isopropyl ester;-   4-Acetoxy-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-ethyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Acetoxy-3-benzylsulfanyl-2-benzylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(1-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid 2-isopropyl-5-methyl-cyclohexyl ester;-   Di-[2-(4-Hydroxy-5-oxo-2-carboxylic acid methyl ester)]-disulfide;    and-   3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester.

In certain embodiments of the invention the dermatologic composition forthe treatment of a dermatological condition additionally comprises anadditional benefit agent, selected from sunscreens, retinoid andderivatives thereof, antioxidants, hydroxyacids, botanical extracts,salicylic acid, benzoylperoxide, antibiotics, antiandrogens,anti-inflammatory agents, vitamins, tocopherol (α-, β-, γ-, δ-) andesters thereof, corticosteroid and mixtures thereof.

Certain embodiments of the invention provide novel and preferredcombinations of substituent groups pendant from the formulae of thedifferent inventions.

In yet another aspect, the invention relates to a method of promoting aproduct by directing a user to apply to the skin a pharmaceutical orcosmetic composition incorporating a compound of any of Formula I, II orIII, or a pharmaceutically acceptable salt thereof, for regulating skincondition, regulating the signs of skin aging, or for treating contactdermatitis, skin irritation, rosacea, acne, psoriasis, age-relateddamage or damage resulting from harmful (UV) radiation or environmentalpollution, stress of fatigue. The invention also entails a productincluding instructions directing the user to apply a composition of theinvention to the skin for regulating skin condition, regulating signs ofskin aging, or for treating contact dermatitis, skin irritation,rosacea, acne, psoriasis, age-related damage or damage resulting fromharmful (UN) radiation or environmental pollution, stress or fatigue,where the composition incorporates a compound of any of Formula I, II,or III or a pharmaceutically or cosmetic acceptable salt thereof.

Excluded from the compositions of matter (but, e.g., not from themethods of use and pharmaceutical formulations) of the present inventionis the compound4-hydroxy-3-methanesulfonyl-2-methanesulfonylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the present specification, the following words and phrasesare generally intended to have the meanings as set forth below, exceptto the extent that the context in which they are used indicatesotherwise.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined below. Itwill be understood by those skilled in the art with respect to any groupcontaining one or more substituents that such groups are not intended tointroduce any substitution or substitution patterns that are stericallyimpractical and/or synthetically non-feasible.

Certain compound, reactant, or reaction parameter abbreviations aredefined as follows:

-   -   “DCM” refers to dichloromethane or methylene chloride    -   “DMF” refers to N, N-dimethyl formamide    -   “Eq.” refers to equivalent.    -   “MeOH” refers to methanol.    -   “TFA” refers to trifluoroacetic acid.

The term “acyl” refers to the groups —C(O)—H, —C(O)-(optionallysubstituted alkyl), —C(O)-(optionally substituted cycloalkyl),—C(O)-(optionally substituted alkenyl), —C(O)-(optionally substitutedcycloalkenyl), —C(O)-(optionally substituted aryl), —C(O)-(optionallysubstituted heteroaryl) and —C(O)-(optionally substituted heterocyclyl).

The term “acyloxy” refers to the moiety —O-acyl, including, for example,—O—C(O)-alkyl.

The term “alkoxy” refers to the groups —O-alkyl, —O-alkenyl,—O-cycloalkyl, —O-cycloalkenyl, and —O-alkynyl. Preferred alkoxy groupsare —O-alkyl and include, by way of example, methoxy, ethoxy, n-propoxy,iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,1,2-dimethylbutoxy, and the like.

The term “substituted alkoxy” refers to the groups —O-(substitutedalkyl), —O-(substituted alkenyl), —O-(substituted cycloalkyl),—O-(substituted cycloalkenyl), —O-(substituted alkynyl) and—O-(optionally substituted alkylene)-alkoxy.

The term “alkyl” refers to a monoradical branched or unbranchedsaturated hydrocarbon chain preferably having from about 1 to 20 carbonatoms, more preferably about 1 to 10 carbon atoms, and even morepreferably about 1 to 6 carbon atoms. This term is exemplified by groupssuch as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,n-hexyl, n-decyl, tetradecyl, and the like.

The term “substituted alkyl” refers to an alkyl group in which 1 or more(up to about 5, preferably up to about 3) hydrogen atoms is replaced bya substituent independently selected from the group: ═O, ═S, acyl,acyloxy, optionally substituted alkoxy, optionally substituted amino,azido, carboxyl, (optionally cycloalkyl, optionally substitutedcycloalkenyl, halogen, hydroxyl, nitro, sulfanyl, sulfinyl, sulfanyl,and sulfonic acid. One of the preferred optional substituents for alkylis hydroxy, exemplified by hydroxyalkyl groups, such as 2-hydroxyethyl,3-hydroxypropyl, 3-hydroxybutyl, 4-hydroxybutyl, and the like;dihydroxyalkyl groups (glycols), such as 2,3-dihydroxypropyl,3,4-dihydroxybutyl, 2,4-dihydroxybutyl, and the like; and thosecompounds known as polyethylene glycols, polypropylene glycols andpolybutylene glycols, and the like

The term “alkylene” refers to a diradical derived from the above-definedmonoradical, alkyl. This term is exemplified by groups such as methylene(—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers [e.g., —CH₂CH₂CH₂—and —CH(CH₃)CH₂—] and the like.

The term “substituted alkylene” refers to a diradical derived from theabove-defined monoradical, substituted alkyl. Examples of substitutedalkylenes are chloromethylene (—CH(Cl)—), aminoethylene (—CH(NH₂)CH₂—),methylaminoethylene (—CH(NHMe)CH₂—), 2-carboxypropylene isomers(—CH₂CH(CO₂H)CH₂—), ethoxyethylene (—CH₂CH₂O—CH₂CH₂—),ethyl(N-methyl)aminoethylene (—CH₂CH₂N(CH₃)CH₂CH₂—),1-ethoxy-2-(2-ethoxy-ethoxy)ethylene —CH₂CH₂O—CH₂CH₂—OCH₂CH₂—OCH₂CH₂—),and the like.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NHR or —NRR where eachR is independently selected from the group: optionally substitutedalkyl, optionally substituted cycloalkyl, optionally substitutedalkenyl, optionally substituted cycloalkenyl, optionally substitutedalkynyl, optionally substituted aryl, optionally substituted heteroaryl,optionally substituted heterocyclyl, acyl, optionally substitutedalkoxy, carboxy and alkoxycarbonyl.

The term “amino acid” or “natural amino acid” refers to any of thetwenty (20) common amino acids as generally accepted in the peptide artand represent L-amino acids unless otherwise designated (with theexception of achiral amino acids such as glycine).

The term “substituted amino acid” refers to an amino acid containing oneor more additional chemical moieties that are not normally a part of theamino acid. Such substitutions can be introduced by a targetedderivatizing agent that is capable of reacting with selected side chainsor terminal residues and via other art-accepted methods. For example,cysteinyl residues most commonly are reacted with .alpha.-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions. Glutaminyl and asparaginyl residues arefrequently deamidated to the corresponding glutamyl and aspartylresidues. Alternatively, these residues are deamidated under mildlyacidic conditions. Other modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or theonylresidues, methylation of the .alpha.-amino groups of lysine, arginine,and histidine side chains (see, e.g., T. E. Creighton, Proteins:Structure and Molecule Properties, W. H. Freeman & Co., San Francisco,pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in someinstances, amidation of the C-terminal carboxyl groups.

The term “aromatic” refers to a cyclic or polycyclic moiety having aconjugated unsaturated (4n+2) π electron system (where n is a positiveinteger), sometimes referred to as a delocalized π electron system.

The term “aryl” refers to an aromatic cyclic hydrocarbon group of from 6to 20 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed (fused) rings (e.g., naphthyl or anthryl). Preferred arylsinclude phenyl, naphthyl and the like.

The term “substituted aryl” refers to an aryl group as defined above,which unless otherwise constrained by the definition for the arylsubstituent, is substituted with from 1 to 5 substituents, andpreferably 1 to 3 substituents, independently selected from the groupconsisting of: ═O, ═S, acyl, acyloxy, optionally substituted alkenyl,optionally substituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, sulfanyl, and sulfonic acid. Preferred arylsubstituents include alkyl, alkenyl, alkoxy, halo, cyano, nitro,trihalomethyl, carboxyalkenyl, and sulfinyl.

The term “aryloxy” refers to the group —O-aryl.

The term “substituted aryloxy” refers to the group —O-(substitutedaryl).

The term “aralkyl” refers to the moiety “-alkylene-aryl” each having themeaning as defined herein. Such aralkyl groups are exemplified bybenzyl, phenethyl, 3-naphthylpropyl and the like.

The term “substituted aralkyl” refers to the moiety “-(optionallysubstituted alkylene)-(optionally substituted aryl)”, each having themeaning as defined herein, where at least one of the aryl or alkylenegroups is substituted, e.g., 4-(N-methyl-pyrrolyl)pentylene.

The term “carbonyl” refers to the di-radical “—C(═O)—”, which is alsoillustrated as “—C(O)—”.

The term “(optionally substituted alkoxy)carbonyl” refers to the groups:—C(O)O-(optionally substituted alkyl), —C(O)O-(optionally substitutedcycloalkyl), —C(O)O-(optionally substituted alkenyl), and—C(O)O-(optionally substituted alkynyl). These moieties are alsoreferred to as esters.

The term “(optionally substituted amino)carbonyl” refers to the group—C(O)-(optionally substituted amino). This moiety is also referred to asa primary, secondary or tertiary carboxamide.

The term “(optionally substituted amino)carbonyloxy” refers to the group—O—C(O)-(optionally substituted amino).

The term “carboxy” or “carboxyl” refers to the moiety “—C(O)OH”, whichis also illustrated as “—COOH”.

The term “cognitive disorders” refers to disorders generallycharacterized by symptoms of forgetfulness, confusion, memory loss,impairment in attention and memory, behavioral and relation disorders,abulia, lack of interest, affective disturbances, and/or, in some casespoor personal care. These symptoms may arise as a result of the generalaging process and/or, from organic brain disease, cerebrovasculardisease, head injury, or developmental or genetic defects. Cognitivedisorders include Alzheimer's disease, senile dementia, anxiety,HIV-related dementia, diabetic neuropathies; depression; Parkinson'sdisease; drug dependency; substance abuse; consciousness disorders,sleeping disorders, disorders of the circadian rhythm, mood disorders,epilepsy; Down's syndrome; Huntington's chorea or disease;stress-related somatic disorders; Pick's disease, Friedreich's ataxia,Creutzfeldt-Jacob disease; disorders associated with panic, phobia orstress.

The term “compound of Formula I or Formula III” is intended to encompassthe furanone derivatives of the invention as disclosed, and/or thepharmaceutically acceptable salts of such compounds or metal complexesthereof. In addition, the compounds of this invention include the ketoand enol tautomers, individual stereochemical isomers (arising from theselection of substituent groups) and mixtures of tautomers and/orisomers.

The term “cosmetics” includes make-up, foundation, and skin careproducts. The term “make-up” refers to products that leave color on theface, including foundations, blacks and browns, i.e., mascara,concealers, eye liners, brow colors, eye shadows, blushers, lip colors,and so forth. The term “foundation” refers to liquid, creme, mousse,pancake, compact, concealer or like products that even out the overallcoloring of the skin. Foundation is typically manufactured to workbetter over moisturized and/or oiled skin. The term “skin care products”refers to products used to treat or otherwise care for, moisturize,improve, or clean the skin. Products contemplated by the phrase “skincare products” include, but are not limited to, adhesives, bandages,toothpaste, anhydrous occlusive moisturizers, antiperspirants,deodorants, powder laundry detergent, fabric softener towels, occlusivedrug delivery patches, nail polish, powders, tissues, wipes, solidemulsion compact, anhydrous hair conditioners medicated shampoos, scalptreatments and the like.

The term “dermatologically-acceptable,” as used herein, means that thecompositions or components thereof so-described are suitable for use incontact with human skin without undue toxicity, incompatibility,instability, allergic response, or the like.

The term “cycloalkyl” refers to non-aromatic cyclic hydrocarbon groupsof having about 3 to 40 (preferably about 4 to 15) carbon atoms having asingle ring or multiple condensed rings. Such cycloalkyl groups include,by way of example, single ring structures such as cyclopropyl,cyclobutyl,

The term “cycloalkyl” refers to non-aromatic cyclic hydrocarbon groupsof having about 3 to 40 (preferably about 4 to 15) carbon atoms having asingle ring or multiple condensed rings. Such cycloalkyl groups include,by way of example, single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ringstructures such as adamantanyl, and the like.

The term “substituted cycloalkyl” refers to a cycloalkyl groupsubstituted with from 1 to 5 substituents, and preferably 1 to 3substituents, independently selected from the group consisting of: ═O,═S, acyl, acyloxy, optionally substituted alkenyl, optionallysubstituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, sulfanyl, and sulfonic acid. A cycloalkyl ringsubstituted with an alkyl group is also referred as “alkylcycloalkyl”.

The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

The term “heteroaryl” refers to an aromatic cyclic hydrocarbon grouphaving about 1 to 40 (preferably from about 3 to 15) carbon atoms andabout 1 to 10 hetero atoms (preferably about 1 to 4 heteroatoms,selected from nitrogen, sulfur, selenium, phosphorus, and/or oxygen)within at least one ring. Such heteroaryl groups can have a single ring(e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinylor benzothienyl. Preferred heteroaryls include pyridyl, pyrryl andfuryl. Other preferred heteroaryls include imidazole, triazole,tetrazole, thiadiazole, oxodiazole, pyrazole, benzoimidazole,benzooxazole, benzoselenazole, and benzothiazole.

The term “substituted heteroaryl” refers to a heteroaryl group asdefined above, which unless otherwise constrained by the definition forthe heteroaryl substituent, is substituted with from 1 to 5substituents, and preferably 1 to 3 substituents, independently selectedfrom the group consisting of: ═O, ═S, acyl, acyloxy, optionallysubstituted alkenyl, optionally substituted alkoxy, optionallysubstituted substituted aryl, optionally substituted aryloxy, azido,carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, sulfanyl, and sulfonic acid.

The term “heteroaralkyl” refers to the moiety “-alkylene-heteroaryl”each having the meaning as defined herein.

The term “substituted heteroaralkyl” refers to the moiety “-(optionallysubstituted alkylene)-(optionally substituted heteroaryl)”, each havingthe meaning as defined herein.

The term “heteroaryloxy” refers to the group —O-heteroaryl.

The terms “heterocycle”, “heterocyclic” and “heterocyclyl” refer to amonoradical, saturated or unsaturated, non-aromatic cyclic hydrocarbongroup having about 1 to 40 (preferably from about 3 to 15) carbon atomsand about 1 to 10 hetero atoms (preferably about 1 to 4 heteroatoms,selected from nitrogen, sulfur, phosphorus, and/or oxygen) within thering. Such heterocyclic groups can have a single ring or multiplecondensed rings. Preferred heterocyclics include morpholino,piperidinyl, and the like.

“Regulating skin condition” includes prophylactically regulating and/ortherapeutically regulating skin condition, including visible and/ortactile discontinuities in skin such as, but not limited to, regulatingvisible and/or tactile discontinuities in the texture of skin, reducingpost-inflammatory hyperpigmentation, regulating non-melanindiscoloration of skin, regulating moisturization and barrier propertiesof skin, atrophy, regulating the elasticity of skin, reducing oily skin,regulating cellulite in skin, regulating pruritus in skin, and promotingwound healing in skin. As used herein, prophylactically regulating skincondition includes delaying, minimizing and/or preventing visible and/ortactile discontinuities in skin. As used herein, therapeuticallyregulating skin condition includes ameliorating, e.g., diminishing,minimizing and/or effacing, discontinuities in skin. Regulating skincondition involves improving skin appearance and/or feel.

“Regulating the signs of skin aging” includes prophylacticallyregulating and/or therapeutically regulating one or more of such signs(similarly, regulating a given sign of skin aging, e.g., lines, wrinklesor pores, includes prophylactically regulating and/or therapeuticallyregulating that sign). As used herein, prophylactically regulating suchsigns includes delaying, minimizing and/or preventing signs of skinaging. As used herein, therapeutically regulating such signs includesameliorating, e.g., diminishing, minimizing and/or effacing signs ofskin aging.

“Signs of skin aging” include, but are not limited to, all outwardvisibly and tactilely perceptible manifestations as well as any othermacro or micro effects due to skin aging. Such signs may be induced orcaused by intrinsic factors or extrinsic factors, e.g., chronologicalaging and/or environmental damage (e.g., sunlight, UV, smoke, ozone,pollutants, stress, etc.). These signs may result from wrinkles,including both fine superficial wrinkles and coarse deep wrinkles, skinlines, facial frown lines, expression lines, rhytides, dermatoheliosis,photodamage, premature skin aging, crevices, bumps, pits, large pores(e.g., associated with adnexal structures such as sweat gland ducts,sebaceous glands, or hair follicles), “orange-peel” skin appearance,dryness, scaliness, flakiness and/or other forms of skin unevenness orroughness; blemishes such as acne, pimples, breakouts; excess skin oilproblems such as over production of sebum, oiliness, facial shine,foundation breakthrough; abnormal desquamation (or hair follicles),“orange-peel” skin appearance, dryness, scaliness, flakiness and/orother forms of skin flakiness, keratoses, hyperkeratinization;inadequate skin moisturization (or hydration) such as caused by skinbarrier damage, environmental dryness; loss of skin elasticity (lossand/or inactivation of functional skin elastin) such as elastosis,sagging (including puffiness in the eye area and jowls), loss of skinfirmness, loss of skin tightness, loss of skin recoil from deformation;non-melanin skin discoloration such as undereye circles, blotching(e.g., uneven red coloration due to, e.g., rosacea), sallowness (palecolor), discoloration caused by telangiectasia or spider vessels;melanin-related hyperpigmented (or unevenly pigmented) skin regions suchas age spots (liver spots, brown spots) and freckles; post-inflammatoryhyperpigmentation such as that which occurs following an inflammatoryevent (e.g., as an such as, but not limited to, that associated withaging or steroid use; other histological or microscopic alterations inskin components such as ground substance (e.g., hyaluronic acid,glycosaminoglycans, etc.), collagen breakdown and structural alterationsor abnormalities (e.g., changes in the stratum corneum, dermis,epidermis, the skin vascular system such as telangiectasia or spidervessels); tissue responses to insult such as itch or pruritus; andalterations to underlying tissues (e.g., subcutaneous fat, cellulite,muscles, trabeculae, septae, and the like), especially those proximateto the skin.

The terms “substituted heterocycle”, “substituted heterocyclic” and“substituted heterocyclyl” refer to a heterocyclyl group as definedabove, which unless otherwise constrained by the definition for theheterocycle, is substituted with from 1 to 5 substituents, andpreferably 1 to 3 substituents, independently selected from the groupconsisting of: ═O, ═S, acyl, acyloxy, optionally substituted alkenyl,optionally substituted alkoxy, optionally substituted alkyl (such astri-halomethyl), optionally substituted alkynyl, optionally substitutedamino, optionally substituted aryl, optionally substituted aryloxy,azido, carboxyl, (optionally substituted alkoxy)carbonyl, (optionallysubstituted amino)carbonyl, cyano, optionally substituted cycloalkyl,optionally substituted cycloalkenyl, halogen, optionally substitutedheteroaryl, optionally substituted heteroaryloxy, optionally substitutedheterocyclyl, optionally substituted heterocyclooxy, hydroxyl, nitro,sulfanyl, sulfinyl, sulfanyl and sulfonic acid.

The term “heterocycloalkyl” refers to the moiety “-alkylene-heterocycle”each having the meaning as defined herein.

The term “substituted heterocycloalkyl” refers to the moiety“-(optionally substituted alkylene)-(optionally substitutedheterocycle)”, each having the meaning as defined herein.

The term “heterocyclooxy” refers to the group —O-heterocycle.

The term “inflammation”, “inflammatory conditions”, or “inflammationconditions” includes but is not limited to muscle fatigue,osteoarthritis, rheumatoid arthritis, inflammatory bowel syndrome ordisorder, skin inflammation, such as atopic dermatitis, contactdermatitis, allergic dermatitis, xerosis, eczema, rosacea, seborrhea,psoriasis, atherosclerosis, thermal and radiation burns, acne, oilyskin, harmful UV damage, keratinization abnormalities, skin irritationincluding retinoid induced irritation, hirsutism, alopecia,dyspigmentation, inflammation due to wounds, scarring or stretch marks,loss of elasticity, skin atrophy and gingivitis.

The term “ischemia” refers to deficiency of blood to an organ or tissuedue to functional constriction or actual obstruction of a blood vessel.Cerebral ischemia, also known as stroke, usually results from theinterruption or reduction of blood and oxygen to the blood vessels ofthe brain; more rarely this may be the result of an hemorrhage. Signs ofstroke include paralysis, slurred speech, general confusion, impairmentof gait, cortical sensory loss over toes, foot and leg, and urinaryincontinence, to name just a few. Many types of heart disease includingcardiac arrhythmias or diseases due to cardiac structural abnormalitiesmay produce cerebral emboli. Atrial fibrillation from any cause,including rheumatic valvular disease, may result in emboli beingproduced which can migrate into the arteries of the brain. Emboliformation and migration can occur as a result of atheroscleroticcardiovascular disease and myocardial infarction. Emboli formation isalso a definite risk for intracardiac surgery and prosthetic valvereplacement. Heart bypass surgery and angioplasty can result in theformation of microemboli which can migrate into the arteries of thebrain and cause a series of occlusions in a number of arteries,resulting in mental impairment. Cerebral embolism is also the principalcomplication in the transplant of artificial hearts. Furthermore, theoverall risk of stroke after any type of general surgery is 0.2 to 1percent. The vegetations of acute and subacute bacterial endocarditiscan give rise to emboli which can occlude a major intracranial artery.Populations at risk of ischemia include but are not limited to patientsscheduled for coronary arterial bypass graft surgery (CABG), patients atrisk for postoperative complications, patients with subarachnoidhemorrhage (SAH), patients with a first or second ischemic stroke,patients with acute ischemic stroke, patients undergoing cardiopulmonaryresuscitation (CPR), patients with temporary lobectomy, patients withdominant hemisphere resection, patients receiving prophylactic brainradiation, patients with closed head trauma with neurological loss,patients with microvascular multi-infarct dementia, patients withhomozygous and heterozygous MELAS (Mitochondrial myopathy,encephalopathy, lactacidosis, stroke); patients with atherosclerotic orprogressive supranuclear palsy disease, patients with symptomatic andasymptomatic Huntington's disease, patients with neonatal asphyxia,patients with meningitis or encephalitis, patients with post herpeticneuropathy, patients with intermittent claudication, patients withspinal cord injury, patients with Huntington's disease, AmyotrophicLateral Sclerosis (ALS) or Friedreich's ataxia, patients with diabeticneuropathy or patients with a disease associated with a hypercoagulablestate secondary to systemic disease, carcinoma, vasoconstriction(including reversible cerebral vasoconstriction, e.g. migraine, trauma,idiopathy), or venous conditions (including dehydration, pulmonaryembolism, pericranial infection, postpartum and postoperative states andsystem cancer).

The term “neurodegenerative disorders” refers to disorders characterizedby a loss of neurons and may or may not include an inflammatory process.Neurodegenerative disorders include stroke, head trauma, cerebralhypoxia, spinal cord injury, senile dementia, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), cerebral amyloid angiopathy,HIV-related dementia, Parkinson's disease, Huntington's disease, priondiseases, myasthenia gravis, Down's syndrome, Creutzfeldt-Jakob disease,diabetic neuropathy, neuropathic pain, encephalitis, meningitis, andDuchenne's muscular dystrophy.

The term “neuroinflammation” or “neuroinflammatory diseases, disordersor conditions” refers to diseases, disorders or conditions characterizedby large numbers of reactive microglia in postmortem brain samples,indicative of an active inflammatory process (McGeer E. G. and McGeer P.L., “Neurodegeneration and the immune system”. Calne D. B., ed.Neurodegenerative Diseases, 1994:277-300). Neuroinflammation refers toinflammation which occurs in response to brain injury or autoimmunedisorders, and has been shown to cause destruction of healthy neuronaland/or cerebral tissue. Neuroinflammation relates to mechanismsimplicated in a broad range of acute and chronic neurodegenerativedisorders, including stroke, head trauma, cerebral amyloid angiopathy,HIV-related dementia, Huntington's disease, prion diseases, meningitis,myelin degradation, Down's syndrome, post-ischemic brain injury,encephalopathy, Parkinson's disease, senile dementia, Alzheimer'sdisease, amyotrophic lateral sclerosis, multiple sclerosis and certaindisorders involving the peripheral nervous system, such as myastheniagravis and Duchenne's muscular dystrophy.

As used herein, “pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The term “pharmaceutically acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thisinvention and which are not biologically or otherwise undesirable. Inmany cases, the compounds of this invention are capable of forming acidand/or base salts by virtue of the presence of amino and/or carboxylgroups or groups similar thereto. Pharmaceutically acceptable baseaddition salts can be prepared from inorganic and organic bases. Saltsderived from inorganic bases, include by way of example only, sodium,potassium, lithium, ammonium, calcium and magnesium salts. Salts derivedfrom organic bases include, but are not limited to, salts of primary,secondary and tertiary amines, such as alkyl amines, dialkyl amines,trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines,tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines,trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl)amines, tri(substituted alkenyl) amines, cycloalkyl amines,di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkylamines, disubstituted cycloalkyl amine, trisubstituted cycloalkylamines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl)amines, substituted cycloalkenyl amines, disubstituted cycloalkenylamine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines,triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroarylamines, heterocyclic amines, diheterocyclic amines, triheterocyclicamines, mixed di- and tri-amines where at least two of the substituentson the amine are different and are selected from the group consisting ofalkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,heteroaryl, heterocyclic, and the like. Also included are amines wherethe two or three substituents, together with the amino nitrogen, form aheterocyclic or heteroaryl group.

Specific examples of suitable amines include, by way of example only,isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine,tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine,purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and thelike.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

The term “sulfanyl” refers to the groups: —S-(optionally substitutedalkyl), —S-(optionally substituted aryl), —S-(optionally substitutedheteroaryl), —S-(optionally substituted heterocyclyl). Preferredsulfanyl groups include, by way of example, methylsulfanyl (—SCH₃),n-(iso-propylsulfanyl) (—SCH(CH₃)₂) and the like.

The term “sulfinyl” refers to the groups: —S(O)-(optionally substitutedalkyl), —S(O)-optionally substituted aryl), —S(O)-(optionallysubstituted heteroaryl), —S(O)-(optionally substituted heterocyclyl).

The term “sulfonyl” refers to the groups: —S(O₂)-(optionally substitutedalkyl), —S(O₂)-optionally substituted aryl), —S(O₂)-(optionallysubstituted heteroaryl), —S(O₂)-(optionally substituted heterocyclyl).

The term “sulfonic acid” refers to the group: —S(O₂)—OH.

The term “tautomers” refers to compounds whose structures differmarkedly in arrangement of atoms, but which exist in easy and rapidequilibrium. The most common kind of tautomerism involves structuresthat differ in the point of attachment of hydrogen. As is well-known inthe art, the 3-hydroxy-5H-furan-2-one group in compounds such as thecompounds of this invention can be in tautomeric equilibrium with thedihydrofuran 2,3-dione group:

For convenience, all the compounds of this invention are shown as havingthe 3-hydroxy-5H-furan-2-one form, but it is to be understood thatcompounds of both tautomeric forms are intended to be within the scopeof the invention.

The term “therapeutically effective amount” refers to that amount of acompound of Formula I that is sufficient to effect treatment, as definedbelow, when administered to a mammal in need of such treatment. Thetherapeutically effective amount will vary depending upon the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the particular compound ofFormula I or Formula III chosen, the dosing regimen to be followed,timing of administration, the manner of administration and the like, allof which can readily be determined by one of ordinary skill in the art.

The term “treatment” or “treating” means any treatment of a disease ordisorder in a mammal, including:

-   -   preventing or protecting against the disease or disorder, that        is, causing the clinical symptoms not to develop;    -   inhibiting the disease or disorder, that is, arresting or        suppressing the development of clinical symptoms; and/or    -   relieving the disease or disorder that is, causing the        regression of clinical symptoms.        It will be understood by those skilled in the art that in human        medicine, it is not always possible to distinguish between        “preventing” and “suppressing” since the ultimate inductive        event or events may be unknown, latent, or the patient is not        ascertained until well after the occurrence of the event or        events. Therefore, as used herein the term “prophylaxis” is        intended as an element of “treatment” to encompass both        “preventing” and “suppressing” as defined herein. The term        “protection,” as used herein, is meant to include “prophylaxis.”

The term “effective amount” means a dosage sufficient to providetreatment for the disorder or disease state being treated. This willvary depending on the patient, the disease and the treatment beingeffected.

Nomenclature

The compounds of the present invention are named and numbered asdescribed below, for example, with reference to Formulae Ia, Ib, Ic, andId.

Formula Ia represents the compound according to Formula I where R¹ is—C(O)OR′ and R′ is ethyl; R² and R³ are each 1H-benzoimidazol-2-yl; R⁴is hydrogen; X is sulfur and Y is sulfur. The compound is shown withoutspecifying stereo configuration. The compound of Formula Ia can benamed:3-(5-chloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5-chloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester.

Formula Ib represents the compound of Formula I where R¹ is —C(O)OR′ andR′ is ethyl; R² and R³ are each a2-amino-4-[1-(carboxymethyl-carbamoyl)-ethylcarbamoyl]-butyric acid; R⁴is hydrogen; X is sulfur and Y is sulfur. The compound is shown withoutspecifying stereo configuration. The compound of Formula Ib can benamed: 3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid. The analogous compound corresponding to Formula Ib where X and Yare each —S(O)₂— can be named: 3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethanesulfonyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethanesulfonylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid.

Formula Ic represents the compound of Formula I where R¹ is —C(O)OR′ andR′ is ethyl; R⁴ is hydrogen; X and Y are sulfur and X—R² taken togetherwith Y—R³ form a hydroxymethyl substituted aliphatic ring. The compoundof Formula Ic can be named:3-hydroxy-6-hydroxymethyl-2-oxo-5,6-dihydro-2H-1-oxa-4,7-dithia-azulene-8a-carboxylicacid ethyl ester

Formula Id represents the compound according to Formula I where R¹ is—C(O)OR′ and R′ is ethyl; R² is 1H-benzoimidazole, Y taken together withR³ is —P(O)(OR′)₂ where R′ is methyl; R⁴ is hydrogen; and X is —S(O)—.The compound of Formula Id can be named:2-(1H-benzoimidazole-2-sulfinylmethyl)-3-(dimethoxy-phosphoryl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester.

In general, the nomenclature used in this Application was generatedusing version 2.1 of the AUTONOM® naming package within the ChemOffice®version 6.0 suite of programs by CambridgeSoft Corp (Cambridge, Mass.).

Synthesis of the Compounds of the Invention

Synthetic Reaction Parameters

The terms “solvent”, “inert organic solvent” or “inert solvent” mean asolvent inert under the conditions of the reaction being described inconjunction therewith. Solvents employed in synthesis of the compoundsof the invention include, for example, methanol, acetone, water,acetonitrile, 1,4-dioxane, dimethylformamide (“DMF”), benzene, toluene,tetrahydrofuran (“THF”), chloroform, methylene chloride (ordichloromethane), diethyl ether, pyridine and the like, as well asmixtures thereof. Unless specified to the contrary, the solvents used inthe reactions of the present invention are inert organic solvents.

The term “q.s.” means adding a quantity sufficient to achieve a statedfunction, e.g., to bring a solution to the desired volume (i.e., 100%).

Unless specified to the contrary, the reactions described herein takeplace at atmospheric pressure within a temperature range from 0° C. to110° C. (preferably from 0° C. to 25° C.; most preferably at “room” or“ambient” temperature, e.g., 20° C.). Further, unless otherwisespecified, the reaction times and conditions are intended to beapproximate, e.g., taking place at about atmospheric pressure within atemperature range of about 0° C. to about 110° C. (preferably from about0° C. to about 25° C.; most preferably at about “room” or “ambient”temperature, e.g., approximately 20° C.) over a period of about 1 toabout 10 hours (preferably about 5 hours). Parameters given in theExamples are intended to be specific, not approximate.

Isolation and purification of the compounds and intermediates describedherein can be effected, if desired, by any suitable separation orpurification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography orthick-layer chromatography, or a combination of these procedures.Specific illustrations of suitable separation and isolation procedurescan be had by reference to the examples hereinbelow. However, asymmetricsynthetic approaches and other equivalent separation or isolationprocedures can, of course, also be used.

Brief Description of Reaction Schemes

Reaction Scheme 1 illustrates the synthesis of the compounds of FormulaI via an aldol addition between pyruvate derivatives, followed by anintramolecular cyclization reaction in the presence of a base.

Reaction Scheme 2 illustrates synthesis of the compounds of Formula Iwhere X—R² taken together with Y—R³ form an optionally substitutedaliphatic or aromatic ring (where X and Y are —S—).

Reaction Scheme 3 illustrates synthesis of the compounds of Formula IIIvia a base catalyzed condensation between a pyruvate derivative and acarbonyl component of another molecule.

Starting Materials

The compound ethyl-3-bromopyruvate is commercially available, e.g., fromAldrich Chemical Company, Milwaukee, Wis. Other reactants, are likewisecommercially available or may be readily prepared by those skilled inthe art using commonly employed methodology.

The starting materials employed in Reaction Scheme 1, Formulae 101 and102, are prepared as described in co-pending U.S. application Ser. No.10/138,937 and 10/138,032, by contacting a halopyruvate (preferablyethyl-3-bromopyruvate) with a precursor of the formula R²—X—H or R³—Y—H,where R² and R³ have the meanings previously described, and where X andY are —N(R′)— or —S—. Such starting materials include:

-   -   an aryl, aralkyl, heteroaryl or heteroaralkyl compound,    -   a nucleoside, amino acid, di-, tri- or tetra-peptide,    -   an aryl-amine, -thiol, -sulfane, -sulfone,    -   an aralkyl-amine, -thiol, -sulfane, -sulfone,    -   a heteroaryl-amine, -thiol, -sulfane, -sulfone, or    -   a heteroaralkyl-amine, -thiol, -sulfane, -sulfone,        and are contacted in an appropriate solvent (such as methanol,        acetone, water, acetonitrile, 1,4-dioxane or DMF), optionally in        the presence of an organic base (such as a tertiary amine or        imidazole). The reaction takes place at a temperature from 0° C.        to 110° C. (preferably 0° C. to 25° C.) for 30 minutes to 15        hours (preferably 3-5 hours), followed by removal of the        solvent(s), isolation and purification to give the corresponding        product of Formulae 101 or 102. Additional isolation and        purification steps well known to those skilled in the art may be        performed, e.g., to provide single stereo isomers and/or        tautomers. Where X taken together with R² or Y taken together        with R³ is —P(O)(OR′)₂ a phosphite precursor (typically a        trialkylphosphite) is contacted with the halopryuvate.

As illustrated in Reaction Scheme 1, Step 1, an aldol addition betweentwo tautomeric pyruvate derivatives of Formulae 101 and 102 (where R^(a)and R^(b) are lower alkyl, preferably ethyl) gives the correspondingcyclized furanone derivative of Formula I/103. In the synthesis of manyof the compounds of Formula I, the groups X—R² and Y—R³ will be the samethe pyruvate derivative(s) of Formulae 101 and 102, which will beemployed as a single reactant (without regard to tautomeric form). Aldoladdition between pyruvate derivatives of Formulae 101 and 102 where R²and R³ contain chiral centers, produces diastereomers. When R² and R³are achiral, it produces a racemic mixture and the resulting compoundhas only one chiral center at the 5-position of the furanone.

In each instance, the product(s) of Reaction Scheme 1, Step 1 will fallwithin the scope of the compounds of the present invention according toFormula I, where R¹ is —C(O)OR′ (R′ being a lower alkyl groupcorresponding to R^(a) or R^(b)) and R⁴ is hydrogen. The compounds ofFormula I where R¹ is —C(O)OR′ (R′ being other than lower alkyl),—C(O)NR′R″ or —CH₂OR′″, or where R⁴ is other than hydrogen, can beprepared as illustrated after Step 1 in Reaction Scheme 1, (for example,by conversion of R¹ lower alkyl ester to another ester, amide, freeacid, alcohol or like moiety) employing reactants and conditions wellknown to those skilled in organic synthesis.

As illustrated in Reaction Scheme 2, Step 1, an optionally substituteddi-thiol of Formula 201 [where R^(c) is optionally one or moresubstituents selected from lower alkyl, hydroxy(lower alkyl), sulfonicacid (lower alkyl), —C(O)OR′, or represents an optionally substitutedaliphatic or aromatic ring] and a halopyruvate of Formula 202 arereacted to give the corresponding di-thiol-bridged di-pyruvate compoundof Formula 203.

As illustrated in Reaction Scheme 2, Step 2, a compound of Formula 203is cyclized to the corresponding compound of Formula I/204 under aldoladdition conditions similar to those employed in Step 1 of ReactionScheme 1.

As illustrated in Reaction Scheme 3, a base catalyzed condensationbetween the pyruvate of Formula 301 wherein R^(a) is a lower alkylgroup, preferably ethyl, and the carbonyl component of Formula 302wherein R^(a), R⁵, R⁶ and R⁷ are as described herein, can give thecorresponding cyclized furanone derivative of Formula III, wherein R⁶ ishydrogen. The compounds of Formula III where R⁸ is other than hydrogen,can be prepared by ways well known in the art, for example, compoundswherein R⁸ is alkyl carbonyl can be prepared by acetylation of thealcohol group in the presence of a base.

Preferred Processes and Last Steps

The preferred process for generating compounds of Formula I or III is asexemplified for the syntheses of Examples 1-34. This process involvespreparation of pyruvate derivatives and the subsequent aldol additionand cyclization between two pyruvate derivatives. The preparation ofpyruvate precursors has already been exemplified in the previousApplications U.S. Ser. Nos. 10/138,937 and 10/138,032. In thisinvention, the preferred process for the preparation of compounds ofFormula I is the aldol addition and the subsequent cyclization betweenthe tautomers, namely the enol and keto forms, of pyruvate derivativesin the presence of a base.

Thus, in one preferred aspect, the pyruvate derivatives are treated witha base.

In another preferred aspect, the cyclized furanone diastereoisomers areseparated using reverse phase chromatography method if the pyruvateprecursors containing chiral center(s).

In yet another preferred aspect, the cyclized furanone racemic mixtureis separated using chiral column chromatography method if the pyruvateprecursors containing achiral centers.

In still another preferred aspect, the stereoisomers are converted intodifferent pharmaceutically acceptable salts either from one salt toanother or from a salt-free entity as exemplified in Examples.

A compound of Formula I or Formula III is contacted with apharmaceutically acceptable acid to form the corresponding acid additionsalt.

A pharmaceutically acceptable acid addition salt of Formula I or FormulaIII is contacted with a base to form the corresponding free base ofFormula I or Formula III.

Preferred Compounds

The compounds of Formula I and of Formula III encompass the furanonederivatives of the invention as disclosed, and/or the pharmaceuticallyacceptable salts of such compounds. In addition, the compounds of thisinvention include the individual stereochemical isomers or tautomers andmixtures thereof, arising from the selection of substituent groups.

Preferred for the compounds, pharmaceutical formulations, methods ofmanufacture and use of the present invention are the followingcombinations and permutations of substituent groups of Formula I(sub-grouped, respectively, in increasing order of preference):

1. X is the same as Y:

-   -   a. Especially where X and Y are —S— or —S(O)—, preferably —S—.        -   i. Preferably where R⁴ is hydrogen.        -   ii. Preferably where R¹ is —C(O)OR′ and R′ is hydrogen or            lower alkyl.        -   iii. Preferably where R² is the same as R³.            -   Particularly where R² and R³ are selected from                optionally substituted alkyl, optionally substituted                heteroaryl, optionally substituted heteroaralkyl, an                optionally substituted amino acid or an optionally                substituted di-, tri- or tetra-peptide, and especially                as further described as preferred below.                2. R¹ is —C(O)OR′; —CH₂OR′″ or —C(O)NR′R″ where R′, R″                and R′″ are hydrogen or lower alkyl    -   a. Preferably R¹ is —C(O)OR′, where R′ is hydrogen or lower        alkyl        -   i. Especially where R⁴ is hydrogen.        -   ii. Especially where R² is the same as R³            -   1. Particularly where R² and R³ are selected from                optionally substituted alkyl, optionally substituted                cycloalkyl, optionally substituted aryl, optionally                substituted aralkyl, optionally substituted heteroaryl,                optionally substituted heteroaralkyl, an optionally                substituted amino acid or an optionally substituted di-,                tri- or tetra-peptide, and especially as further                described as preferred below.                3. R⁴ is hydrogen.                4. R² is the same as R³:    -   a. Especially where R² and R³ are selected from optionally        substituted alkyl, optionally substituted cycloalkyl, optionally        substituted aryl, optionally substituted aralkyl, optionally        substituted heteroaryl, optionally substituted heteroaralkyl, an        optionally substituted amino acid or an optionally substituted        di-, tri- or tetra-peptide.        -   i. Preferably where R² and R³ are optionally substituted            heteroaryl, optionally substituted heteroaralkyl, optionally            substituted aryl or optionally substituted aralkyl.            -   Particularly where optionally substituted heteroaryl or                optionally substituted heteroaralkyl is selected from:                optionally substituted 4-pyridinyl, optionally                substituted 2-pyridinyl, optionally substituted                1H-benzoimidazol-2-yl, optionally                substituted-1H-benzothiazol-2-yl, optionally substituted                benzooxazole-2-yl; optionally substituted                benselenazol-2-yl; optionally substituted                1H-[1,2,4]triazol-3-yl, optionally substituted                2H-[1,2,4]triazol-3-yl, optionally substituted                4H-[1,2,4]triazol-3-yl, optionally substituted                [1,3,4]oxadiazol-2-yl, optionally substituted                [1,2,4]thiadiazol-5-yl, optionally substituted                [1,3,5]thiadiazol-2-yl optionally substituted                4,5-dihydro-thiazol-2-yl, optionally substituted                1H-pyrazolo[3,4-d]pyrimidin-6-yl, optionally substituted                1H-imidazol-2-yl, optionally substituted quinolyn-4-yl,                optionally substituted pyrimidin-4-yl, optionally                substituted pyrimidin-2-yl, optionally substituted                2H-chromen-7-yl, optionally substituted furan-2-yl-lower                alkyl, and optionally substituted                3,4-dihydro-quinazolin-2-yl, and wherein the                substitutents are selected from (C₁-C₈)alkyl,                (C₁-C₈)alkenyl, halogen, haloalkyl, acyl, sulfonic acid,                sulfonyl, amino, mono- or di-substituted amino, aryl,                carboxy, carboxyvinyl, ester, amide, hydroxy, and                alkoxy.                -   a. More preferably where optionally substituted                    heteroaryl or heteroaralkyl is selected from                    optionally substituted benzyl; optionally                    substituted 1-H-benzoimidazol-2-yl; optionally                    substituted benzothiazol-2yl; optionally substituted                    benzooxazol-2-yl; optionally substituted                    benzosenlenazol-2-yl; optionally substituted                    furan-2-yl-lower alkyl; optionally substituted                    thiazol-2-yl; optionally substituted                    1H-imidazol-2-yl; optionally substituted                    pyridine-2-yl; optionally substituted                    pyrimidin-2-yl; optionally substituted                    quinolinin-4-yl; optionally substituted                    [1,3,4]oxadiazol-2-yl; optionally substituted                    2H-[1,2,4]-triazol-3-yl; and optionally substituted                    [1,3,4]thiadiazole-2-yl; and wherein the                    substituents are selected from (C₁-C₈)alkyl,                    (C₁-C₈)alkenyl, halogen, haloalkyl, acyl, sulfonic                    acid, sulfonyl, amino, mono- or di-substituted                    amino, aryl, carboxy, carboxyvinyl, ester, amide,                    hydroxy, and alkoxy.                -   b. Most preferably 1-H-benzoimidazol-2-yl;                    benzothiazol-2-yl; 5-methoxy-benzothiazol-2-yl;                    6-nitro-benzothiazol-2-yl; benzooxazol-2-yl;                    5-amino-[1,3,4]thiadiazol-2-yl; furan-2-ylmethyl;                    pyridin-4-yl; 5-phenyl-[1,3,4]oxadiazol-2-yl;                    pyrrolidine-1-carbothioyl;                    4-(2-methoxycarbonyl-vinyl)-phenyl;                    4-trifluoromethyl-pyrimidin-2-yl;                    4-methyl-pyrimidin-2-yl; and pyrimidin-2-yl.                -    Especially wherein R¹ is —C(O)OR′, R′ is selected                    from hydrogen and                -    (C₁-C₈)alkyl; and R⁴ is hydrogen.                -    Especially wherein R¹ is —CH₂OR′″; R′″ is selected                    from hydrogen and (C₁-C₈)alkyl; and R⁴ is hydrogen.                -    Especially wherein R¹ is —C(O)NR′R″; R′ and R″ are                    selected from hydrogen, (C₁-C₈)alkyl and                    hydroxy(C₁-C₈)alkyl; and R⁴ is hydrogen.        -   ii. Preferably where R² and R³ are optionally substituted            di-, tri- or tetra-peptides. Particularly            (4-amino-4-carboxyburyrylamino-2-(carboxymethyl-carbamoyl)-ethyl.        -   iii. Preferably where R² and R³ are optionally substituted            alkyl or optionally substituted cycloalkyl.            -   Particularly R² and R³ are selected from                (C₃-C₈)cycloalkyl, butyl,                2-acetylamino-2-methoxycarbonyl-ethyl,                3-(2-carboxy-pyrrolidin-1-yl)-2-methyl-3-oxo-propyl,                2-carboxy-2-isobutyrylamino-ethyl,                2-methoxycarbonyl-ethyl, 3-ethoxycarbonylmethyl;                methoxycarbonylmethyl, carboxymethyl-carbamoyl-ethyl,                dimethylthiocarbamoyl, isobutyl,                2-hydroxy-1-methyl-propyl, 2,3-dihydroxy-propyl, and                allyl.        -   iv. Preferably where R² and R³ are optionally substituted            aryl or optionally substituted aralkyl,            -   1. Particularly where optionally substituted aryl or                optionally substituted aralkyl are selected from                optionally substituted phenyl, optionally substituted                naphthyl or optionally substituted benzyl.                -   a. More preferably where optionally substituted aryl                    or optionally substituted aralkyl are selected from:                    4-methoxy-benzyl; 2,4-dichloro-benzyl;                    2-chloro-6-fluoro-benzyl; 4-fluoro-benzyl; benzyl;                    2-chloro-phenyl; and 2-chloro-4-fluoro-phenyl.                    5. X—R² taken together with Y—R³ forms an optionally                    substituted aliphatic or aromatic ring.    -   a. Especially an optionally substituted dithia-cyclohexene,        optionally substituted dithia-cycloheptene,        7,8-dihydro-6H-5,9-dithia-benzocycloheptene,        -   i. Particularly            1,4-dichloro-7,8-dihydro-6H-5,9-dithia-benzocycloheptene,            2,3-dimethyl-[1,4]dithiepane, or [1,3]dithiane-2-thione.            6. Metal complex where the metal is selected from divalent            copper, manganese and zinc    -   a. Especially where the metal is selected from Cu²⁺Cl₂, Mn²⁺Cl₂,        and Zn²⁺Cl₂,        -   i. Particularly with a compound of Formula I wherein XR² and            YR³ are both benzoimidazol-2-ylsulfanyl.

Of the compounds where X—R² and/or Y—R³ are represented by Formula II,preferred are those compounds the substituents of which are selectedfrom the following groups:

-   -   R^(2.1) is —C(O)—O—R′ where R′ is hydrogen or lower alkyl,        especially ethyl;    -   R^(2.2) is hydrogen;    -   R^(2.3) is —CH₂—S—;    -   R^(2.4) is hydrogen, optionally substituted lower alkyl,        optionally substituted aralkyl, optionally substituted        heteroaralkyl;    -   R^(2.5) is hydrogen or lower alkyl, especially hydrogen; and/or    -   k, m and n are respectively: 0,2,1; 1,0,1; or 2,0,1.

Also preferred for the compounds, pharmaceutical formulations, methodsof manufacture and use of the present invention are the followingcombinations and permutations of substituent groups of Formula III(sub-grouped, respectively, in increasing order of preference):

-   1. Y′ is —S—, —S(O)— or —S(O)₂—; especially where Y′ is —S—.    -   i. Preferably where R⁷ is selected from optionally substituted        phenyl, optionally substituted naphthalenyl, optionally        substituted benzyl, optionally substituted        1-H-benzoimidazol2-yl, optionally substituted benzothiazole-2yl,        optionally substituted benzooxazole-2-yl, optionally substituted        furan-2-yl-lower alkyl, optionally substituted thiazol-2-yl,        optionally substituted 1H-imidazol-2-yl, optionally substituted        pyridine-2-yl, optionally substituted pyrimidin-2-yl, optionally        substituted quinolinin-4-yl; optionally substituted        [1,3,4]oxadiaazol-2-yl, optionally substituted        2H[1,2,4]-triazol-3-yl.        -   a. more preferably where R⁷ is selected from benzyl;            4-fluorobenzyl; 1-H-benzoimidazol-2-yl;            5-methyl-1-H-benzoimidazol2-yl; benzothiazole-2yl;            5-chloro-benzothiazole-2yl; and 4-phenyl-thiazol-2-yl.    -   ii. Preferably where R⁸ is hydrogen    -   iii. Preferably where R⁵ is —C(O)OR^(a) or —C(O)R^(c), R^(a) is        selected from hydrogen, (C₁-C₅)alkyl, and        (C₁-C₈)alkyl-(C₃-C₈)cycloalkyl, and R^(c) is selected from        hydrogen, (C₁-C₈)alkyl, and aryl,        -   a. More preferably where R⁶ is hydrogen or —C(O)OR^(a) and            R^(a) is selected from hydrogen and (C₁-C₅)alkyl.    -   iv. Preferably where R⁵ is —C(O)R^(c), and R^(c) is selected        from hydrogen, (C₁-C₈)alkyl, and aryl, and R⁶ is hydrogen.-   2. R⁵ is —C(O)OR^(a) or —C(O)R^(c), R^(a) is selected from hydrogen,    (C₁-C₈)alkyl, and (C₁-C₈)alkyl-(C₃-C₈)cycloalkyl, and R^(c) is    selected from hydrogen, (C₁-C₈)alkyl, and aryl.    -   i. Especially where R⁸ is hydrogen.    -   ii. Especially where R⁷ is selected from optionally substituted        phenyl, optionally substituted naphthalenyl, optionally        substituted benzyl, optionally substituted        1-H-benzoimidazol2-yl, optionally substituted benzothiazole-2yl,        optionally substituted benzooxazole-2-yl, optionally substituted        furan-2-yl-lower alkyl, optionally substituted thiazol-2-yl,        optionally substituted 1H-imidazol-2-yl, optionally substituted        pyridine-2-yl, optionally substituted pyrimidin-2-yl, optionally        substituted quinolinin-4-yl; optionally substituted        [1,3,4]oxadiaazol-2-yl, optionally substituted        2H-[1,2,4]-triazol-3-yl.        -   a. Preferably where R⁷ is selected from benzyl;            4-fluorobenzyl; 1-H-benzoimidazol-2-yl;            5-methyl-1-H-benzoimidazol2-yl; benzothiazole-2yl;            5-chloro-benzothiazole-2yl; and 4-phenyl-thiazol-2-yl.-   3. R⁵ and R⁶ taken together with the atom to which they are attached    form a ring optionally incorporating one or more additional    heteroatoms chosen from N, O or S and optionally substituted with    one or more substitutents selected from the group consisting of    optionally substituted lower alkyl, halo, carboxy, and oxo;    -   a. Especially a pyrimidine 2,4,6-trione ring or a cyclohexanone        ring.-   4. R⁵ and R⁷ together with the atoms to which they are attached form    a ring optionally incorporating one or more additional heteroatoms    chosen from N, O or S and optionally substituted with one or more    substituents selected from the group consisting of optionally    substituted lower alkyl, halo, carboxy and oxo;    -   a. Especially a 3-methyl-thiomorpholin-3-ol ring and the        compound formed is        1,4-dihydro-4-methyl-3a,4-dihydro-3-oxa-10-thia-4a,9-diaza-cyclopenta[b]fluoren-2-one.

The preferred compounds include the following, as well as theirstereoisomer, tautomers, salts, and mixtures thereof:

-   4-(Benzothiazol-2-ylsulfanyl)-3-hydroxy-1-oxa-spiro[4.5]dec-3-ene-2,6-dione;-   Methanesulfonic acid    3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethyl    ester;-   3-(Benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   3-[9-(3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-2-methyl-9H-purin-6-ylsulfanyl]-2-[9-(3,4-dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl)-2-methyl-9H-purin-6-ylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(2-methyl-propane-1-sulfonyl)-2-(2-methyl-propane-1-sulfonylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(9H-purin-6-ylsulfanyl)-2-(9H-purin-6-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid methyl ester;-   4-Hydroxy-5-oxo-3-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-2-methyl-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-(4-methyl-piperazine-1-carbonyl)-5H-furan-2-one;-   4-Hydroxy-5-oxo-3-(3-sulfo-propylsulfanyl)-2-(3-sulfo-propylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester, as hydrochloric acid salt; and-   3-[3-(2-Carboxy-pyrrolidin-1-yl)-2-methyl-3-oxo-propylsulfanyl]-2-[3-(2-carboxy-pyrrolidin-1-yl)-2-methyl-3-oxo-propylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

The more preferred compounds include the following, as well as theirstereoisomers, tautomers, salts, and mixtures thereof:

-   4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-5-thiazol-2-yl-5H-furan-2-one;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4.5]dec-3-ene-2,6,8,10-tetraone;-   Phosphoric acid    mono-[3-(benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethyl]ester;-   4-(Benzothiazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4.5]dec-3-ene-2,6,8,10-tetraone;-   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2,5-dihydro-furan-2-carboxylic    acid;-   3-[(4-(2-Carboxy-vinyl)-phenylsulfanyl]-2-[4-(2-carboxy-vinyl)-phenylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   Di-[2-(4-Hydroxy-5-oxo-2-carboxylic acid methyl ester)]-disulfide;-   4-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-5-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(9H-purin-6-ylsulfanyl)-2-(9H-purin-6-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(5-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   4-Hydroxy-3-(1H-imidazol-2-ylsulfanyl)-2-(1H-imidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Diethylamino-ethylsulfanyl)-2-(2-diethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-2-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid methyl ester;-   4-Hydroxy-3-(2-methoxycarbonyl-ethylsulfanyl)-2-(2-methoxycarbonyl-ethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-methoxycarbonylmethylsulfanyl-2-methoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methoxy-1H-benzoimidazol-2-ylsulfanyl)-2-(5-methoxy-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-nitro-1H-benzoimidazol-2-ylsulfanyl)-2-(5-nitro-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   4-Hydroxy-5-oxo-3-p-tolylsulfanyl-2-p-tolylsulfanylmethyl-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

The most preferred compounds include the following as well as theirstereoisomers, tautomers, salts, and mixtures thereof:

-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-thiazol-2-yl-5H-furan-2-one;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid (2-hydroxy-ethyl)-amide;-   4-(Benzothiazol-2-ylsulfanyl)-5-benzoyl-3-hydroxy-5H-furan-2-one;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   Dimethylamino-acetic acid    3-(benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethyl    ester;-   4-(5-Chloro-benzothiazol-2-ylsulfanyl)-5-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   4-(Benzothiazol-2-ylsulfanyl)-5-(benzothiazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   3-(5,6-Dichloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5,6-dichloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   4-(Furan-2-ylmethylsulfanyl)-5-(furan-2-ylmethylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   5-Acetyl-4-(benzothiazol-2-ylsulfanyl)-3-hydroxy-5H-furan-2-one;-   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Furan-2-ylmethanesulfonyl)-2-(furan-2-ylmethanesulfonylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Benzylsulfanyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic acid    ethyl ester;-   4-Hydroxy-3-methylsulfanyl-2-methylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(Benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-Hydroxy-5,6-dimethyl-2-oxo-5,6-dihydro-2H-1-oxa-4,7-dithia-azulene-8a-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid 2-isopropyl-5-methyl-cyclohexyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(1-methyl-1H-imidazol-2-ylsulfanyl)-2-(1-methyl-1H-imidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclopentylsulfanyl-2-cyclopentylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-isobutylsulfanyl-2-isobutylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(thiazol-2-ylsulfanyl)-2-(thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzooxazol-2-ylsulfanyl)-2-(benzooxazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   4-Hydroxy-5-oxo-3-(pyridin-4-ylsulfanyl)-2-(pyridin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   5,8-Dichloro-3-hydroxy-2-oxo-2H-1-oxa-4,9-dithia-benzo[f]azulene-10a-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanyl)-2-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-amino-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(3-Amino-[1,2,4]thiadiazol-5-ylsulfanyl)-2-(3-amino-[1,2,4]thiadiazol-5-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethanesulfonyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethanesulfonylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   R-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)    ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   S-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)    ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   R-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)    ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(2-Acetylamino-2-methoxycarbonyl-ethylsulfanyl)-2-(2-acetylamino-2-methoxycarbonyl-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   (S)-3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   (R)-(3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

Other most preferred compounds include the following as well as theirstereoisomers, tautomers, salts, and mixtures thereof:

-   3-(2-Chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   4-(Benzooxazol-2-ylsulfanyl)-5-(benzooxazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(Benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzooxazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid methyl ester;-   4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-Dimethyl-propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-Dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Butylsulfanyl-2-butylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methoxy-benzylsulfanyl)-2-(4-methoxy-benzylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methoxy-phenylsulfanyl)-2-(4-methoxy-phenylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-oxy-pyridin-2-ylsulfanyl)-2-(1-oxy-pyridin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methyl-pyrimidin-2-ylsulfanyl)-2-(4-methyl-pyrimidin-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

Another preferred sub-group of compounds (including single tautomers,single steroisomers and mixtures of tautomers and/or stereoisomers, andthe pharmaceutically or cosmetically acceptable salts) employed ascompound for use in cosmetic compositions of the present invention is:

-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(2-chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester-   3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid 2-isopropyl-5-methyl-cyclohexyl ester;-   Di-[2-(4-Hydroxy-5-oxo-2-carboxylic acid methyl ester)]-disulfide;-   3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4.5]dec-3-ene-2,6,8,10-tetraone;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid isopropyl ester; and-   4-Hydroxy-3-(1-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

Utility, Testing and Administration

General Utility

Compounds of the present invention are useful in treating a number ofdisorders, particularly those characterized by oxidative stress and/orinflammation. In particular, compounds of the present invention can beused in the treatment of cerebral ischemia (“stroke” most often causedby thrombosis, vasoconstriction and embolism), myocardial ischemia(including chronic stable angina, angina pectoris, unstable angina andPrinzmetal's angina, silent ischemia, reinfarction, reocclusion,restenosis, myocardial infarction and other forms of heart disease),diabetes, renal disease, pre-menstrual syndrome, asthma, cardiopulmonaryinflammatory disorders, chronic heart failure, rheumatoid arthritis,muscle fatigue, irritable bowel syndrome, inflammatory bowel disease,intermittent claudication and for the preservation of allograft tissuefor transplantation. Compounds of the present invention are also usefulin treating conditions falling with the group of dermatologicconditions, in particular prevention and protecting skin tissue againstage-related damage or damage resulting from insults such as harmfulultraviolet (UV) radiation, stress and fatigue, and in the treatment ofcontact dermatitis, skin irritation, skin pigmentation, rosacea,psoriasis, or acne. Compounds of the present invention are also usefulin treating a number of disorders associated with neuroinflammation,cognition, and neurodegeneration, particularly Friedreich's ataxia,epilepsy, Parkinson's disease, Alzheimer's disease, and senile dementia.

Without subscribing to a particular theory or mechanism of action,compounds of the invention may target certain enzymes known as“oxidoreductases” that function widely across a variety of physiologicalprocesses. In particular, oxidoreductases catalyze reactions in whichtwo molecules interact so that one molecule is oxidized and the other isreduced, with a molecule of water entering the reaction. Some of themost important oxidoreductases include, dehydrogenases (includingubiquinone), reductases, peroxidases, nitric acid synthases, cholesteroloxidases, acyl-coenzyme A dehydrogenases and reductases, hydroxylases,and the like. These enzymes play roles in such essential processes asdigestion, signal transduction, maintenance of ionic homeostasis, andthe like. Alterations in oxidoreductases are thought to account for asmany as 3% of all known human genetic diseases. In addition to thediseases and disorders listed above, abnormalities in oxidoreductaseactivity may underly such disorders as congestive heart failure,respiratory chain defects (e.g., abnormalities associated with enzymesof the respiratory chain), glycogen storage disease, rheumatoidarthritis, amyotrophic lateral sclerosis (ALS), chronic alcohol liverdamage (CALD), Refsum's disease (hereditary neuropathy), Crohn'sdisease, Eales Disease (pervasculitis), prion diseases, Huntington'sDisease, cataracts, multiple sclerosis, acute respiratory distresssyndrome (ARDS), Zellweger Syndrome (peroxisomal biogenesis), polycysticovary syndrome, Alpers Syndrome (encephalopathy),steatosis/steatohepatitis, metabolic syndrome, diabetes, asthma, Fanconianemia, retinopathy, age-related macular degeneration (AMD),pre-eclampsia, cholestatic liver disease, end-stage renal disease,Creutzfeldt-Jakob Disease (CJD), and sickle cell disease.

The compounds, formulations and methods of the present invention areuseful in treating a number of dermatological condition, including, butnot limited to prevention and protecting skin tissue against age-relateddamage or damage resulting from insults such as harmful ultraviolet(UV), radiation, stress and fatigue. Such compounds, formulations andmethods are likewise useful, e.g. in treating contact dermatitis, skinirritation, rosacea, acne and psoriasis (including scalp psoriasis), andre amenable to topical application for hair care and treatments of thescalp, for example by incorporation in medicated shampoos, anhydroushair conditioners and the like.

For example, exposure to sunlight can pose a number of hazards to theskin. The major short-term hazard of prolonged exposure to sunlight iserythema, i.e., sunburn, which primarily results from UVB radiationhaving a wavelength of from about 290 nm to about 320 nm. Over the longterm, however, such prolonged exposure can often cause malignant changesin the skin surface to occur. Epidemiologic studies demonstrate a strongrelationship between sunlight exposure and human skin cancer. Anotherlong-term hazard of ultraviolet radiation is premature aging of theskin, which is primarily caused by UVA radiation having a wavelength offrom about 320 nm to about 400 nm. This condition is characterized bywrinkling and pigment changes of the skin, along with other physicalchanges such as cracking, telangiectasis, solar dermatoses, ecchymoses,and loss of elasticity. Individuals, particularly those havinglight-skin who burn easily and tan poorly, who have had a great deal sunexposure in childhood can show the following gross cutaneous alterationsin later adult life: wrinkling, leatheriness, yellowing, looseness,roughness, dryness, mottling (hyperpigmentation) and variouspremalignant growths (often subclinical). These cumulative effects ofsunlight are often referred to as “photoaging”. Although the anatomicaldegradation of the skin is most advanced in the elderly, the destructiveeffects of excessive sun exposure are already evident by the seconddecade. Serious microscopic alterations of the epidermis and dermisoccur decades before these become clinically visible. Wrinkling,yellowing, leatheriness and loss of elasticity are very late changes.

Other skin conditions that may benefit from the methods of the presentinvention include, but are not limited to, diaper rash, a common form ofcontact dermatitis and irritation occurring in infants, as well asadults, who wear diapers. U.S. Pat. No. 6,211,186, incorporated hereinby reference, describes possible etiologies and methods of treating thiscondition. It is generally thought that one or more fecal and lipolyticenzymes, as well as ammonia, bacteria, urine pH, overhydration andCandida albicans may be involved in the onset of skin irritation andinflammation associated with diaper rash. It is also likely thatphysiological responses of the skin to the irritants, such as productionof cytokines by keratinocytes, contribute to the ensuing appearance oferythema, papules, scaling and ulceration characteristic of thecondition. In addition, compositions and methods of the presentinvention may be useful in treating acne, a skin condition characterizedby a profound inflammatory component.

The compositions of the present invention are also useful for regulatingskin condition, including visible and/or tactile discontinuities in skin(especially the skin surface; such discontinuities are generallyundesired). Such discontinuities may be induced or caused by internaland/or external factors, and include the signs of skin aging describedherein.

The compositions of the present invention are useful for regulatingsigns of skin aging, especially visible and/or tactile discontinuitiesin skin texture associated with aging. It is to be understood that thepresent invention is not to be limited to regulation of the “signs ofskin aging” that arise due to the above-mentioned mechanisms associatedwith skin aging, but is intended to include regulation of such signsirrespective of their mechanism of origin.

Testing

This section describes how compositions incorporating compositions ofthe present invention are selected, using in vitro and/or in vivo animalmodels, for example, and used as therapeutic interventions in theexemplary indications, i.e., stroke, chronic heart failure, myocardialinfarction, and Alzheimer's disease.

Insults to the brain that disrupt its blood supply, as in ischemia, orits oxygen supply, as in hypoxia (low oxygen) or anoxia (no oxygen),rapidly cause neuronal imbalance leading to cell death (Flynn, C. J., etal., 1989, in G. Siegel et al., (Eds), Basic Neurochemistry, RavenPress, NY). Investigations into the cellular and molecular mechanismsthat lead to neuronal damage and inflammation associated with varioustypes of brain ischemia can be carried out using in vitro model systems,such as primary cell cultures, that retain the metabolic characteristicsof neurons in vivo. The use of such cell-based models has led toadvances in identification of biochemical mechanisms leading to neuronaldeath in conditions such as anoxia, hypoglycemia, excitotoxicity, andexposure to reactive oxygen species. Neuronal cell lines such as thepheochromocytoma cell line, PC12, are also useful models for studyingthe effects of oxidative stress on the structure and function ofneuron-specific proteins that are expressed in the cell lines. As manyneuronal cell lines do not express all the properties of genuineneurons, primary neuronal cultures are now widely used as in vitromodels in which to discern the processes that occur in intact brain.

In vitro models of ischemia approximate oxygen and glucose deprivationthat mimic in vivo conditions, for example, by placing neuronal culturesinto large anaerobic or hypoxic chambers and exchanging culture mediumwith de-oxygenated and defined ionic composition media. The toxicoverstimulation of neuronal glutamate receptors, especiallyN-methyl-D-aspartate (NMDA) receptors, contributes to hypoxic-ischemicneuronal injury (Choi, D. M., 1988, Neuron 1: 623-634), ischemicinduction of reactive oxygen species (ROS) (Watson, B. D., et al., 1988,Ann NY Acad Sci., 59: 269-281), excessive calcium influx (Grotta, J. C.,1988, Stroke 19: 447-454), arachidonic acid increase (Siesjo, B. K.,1981, J. Cereb. Blood Flow Metab. 1: 155-186) and DNA damage (MacManus,J. P., et al., 1993, Neurosci. Lett., 164: 89-92), each causing acascade of neurodegeneration.

Primary embryonic hippocampal neuronal cells are widely recognized asuseful in models of neuronal function. The hippocampus is a source of arelatively homogenous population of neurons with well-characterizedproperties typical of central nervous system (CNS) neurons in general.Pyramidal neurons, the principal cell type in the hippocampus, have beenestimated to account for 85% to 90% of the total neuronal population(Banker and Goslin, 1998, Culturing Nerve Cells, 2^(nd) edition. The MITPress, Cambridge, Mass.). The hippocampus also exhibits a remarkablecapacity for activity-dependent changes in synaptic function, such aslong-term potentiation (Hawkins R D, Kandel E R, Siegelbaum S A. (1993)Learning to modulate transmitter release: themes and variations insynaptic plasticity [review], Ann. Rev Neurosci. 16:625-665.).

In experiments carried out in support of the present invention accordingto methods detailed in the Examples, anoxia/ischemia was induced inprimary cultures of hippocampal neuronal cells, and compounds weretested for their ability to prevent cell death. Certain compounds foundto have activity in such in vitro assays are then further tested in oneor more animal models of cerebral ischemia (“stroke”), such as themiddle cerebral artery occlusion (MCAO) model in rats.

Briefly, primary cultures of hippocampal neurons are used to testcompounds for activity in neuronal protection. Hippocampal cultures aretypically prepared from 18- to 19-day fetal rats. At this age, thegeneration of pyramidal neurons, which begins in the rat at about E15,is essentially complete. The brain tissue at this stage is relativelyeasy to dissociate, the meninges are removed readily, and the number ofglial cells still is relatively modest (Park L C, Calingasan N Y, UchidaK, Zhang H, Gibson G E. (2000). Metabolic impairment elicits brain celltype-selective changes in oxidative stress and cell death in culture. JNeurochem 74(1): 114-124).

In order to evaluate the activity of compounds of the present invention,a test compound is assessed for its ability to protect cells against oneor more standard stressors, including hypoxia, as detailed in theExamples. In general, desirable therapeutic compound candidates areeffective in this model at concentrations less than about 1 mM and evenmore preferably, less than about 100 μM. By effective, it is meant thatsuch compounds protect at least 20%, preferably 30%, more preferably 40%and even more preferably 50% or more of the cells tested fromstressor-induced death. By way of example, compounds that are effectivein providing protection over a concentration a range of about 1 to 1000μM would be expected to provide neuroprotection in vivo. Since precisevalues may vary depending upon the specific conditions under which theneuroprotective cell assay is carried out, it is the intent of thepresent disclosure to provide the foregoing criteria as guidance in theform of a benchmark against which to compare subsequently testedcompounds, rather than to provide absolute concentrations at which thecompounds of the present invention are considered to be effective.Typically, compounds that are found to be neuroprotective in such invitro cell systems are then further tested in an in vivo animal model ofneuroprotection, such as the rat middle cerebral artery occlusion modeldescribed below, or other appropriate models such as are well known inthe art.

Cerebral ischemic insults are modeled in animals by occluding vesselsto, or within, the cranium (Molinari, G. F., 1986, in H. J. M. Barnett,et al., (Eds) Stroke: Pathophysiology, Diagnosis and Management, Vol. 1,Churchill Livingstone, N.Y.). The rat middle cerebral artery occlusion(MCAO) model is one of the most widely used techniques to inducetransient focal cerebral ischemia approximating cerebral ischemic damagein humans, e.g., those who suffer from a stroke. The middle cerebralartery used as the ischemic trigger in this model is the most affectedvessel in human stroke. The model also entails a period of reperfusion,which typically occurs in human stroke victims. MCAO involving atwo-hour occlusion has been found to produce the maximum size ofcortical infarction obtainable without increased mortality attwenty-four hours.

Briefly, a nylon filament is implanted into the right carotid artery ofthe rat. To effect occlusion, the rat is anesthetized, and the filamentis advanced into the internal carotid artery 18-20 mm from the point ofbifurcation of internal and external arteries and a suture is tightlyligated around the filament for a period of two hours. Two hours postocclusion, animals are re-anesthetized, and the filament is removed, toallow reperfusion for the remainder of the experiment. Test drugs can beadministered any time during this process—before, during or afterocclusion, and can be administered by one or more of a variety of means,including but not limited to intracerebroventricular (ICV) infusion,intravenous (IV) infusion, intraperitoneal (IP) administration, as wellas enteral administration (e.g., gavage). Animals are maintainednormothermic during the experiment, as described in the Examples. At apre-determined time following occlusion and reperfusion, animals aresacrificed and their brains are removed and processed for assessment ofdamage as measured by infarct volume. In general, compounds areconsidered to have activity in this model, if they provide a significantreduction in total infarct volume at a dose that is less than about 10mg/kg, preferably less than 1 mg/kg, more preferably less than 100 μg/kgand even more preferably less than about 1 μg/kg, when administered ICVor IV. By significant reduction of total infarct volume is meant areduction of at least 20%, preferably at least 30%, more preferably atleast 40%, and even more preferably about 50%, compared to controlvalues.

Further validation of efficacy in neuroprotection can be assessed infunctional tests, such as the grip strength test or the rotorod test.Animals treated with compounds that show neuroprotection maintain theirpre-MCAO grip strength values after MCAO, as compared to untreatedanimals, which showed a significant reduction in grip strength,indicating loss of sensorimotor function. Likewise, animals treated withcompounds that show neuroprotection also maintained their pre-MCAOrotorod activity scores after MCAO, as compared to untreated animals,which showed a significant reduction in rotorod scores, indicating lossof sensorimotor function at higher brain levels.

Similarly, primary cultures of myocytes can be used to test compounds invitro for ability to provide protection against heart damage, resultingfor example from myocardial ischemia or congestive heart failure.Preparation of myocardiocytes from neonatal rats is described in theExamples. Such cells are typically used to study molecular models ofmyocardial ischemia (Webster, K A, Discher, D J & Bishopric, N H. 1995.J. Mol. Cell Cardiol. 27:453-458; Camilleri, L, Moins, N, Papon, J,Maublant, J, Bailly, P, de Riberolles, C & Veyre, A. 1997. Cell Biol. &Toxicol. 13:435-444; Bielawska, A E, Shapiro, J P, Jiang, L, Melkonyan,H S, Piot, C, Wolfe, C L, Tomei, L D, Hannun, Y A & Umansky, S R. 1997.Am. J. Pathol. 151:1257-1263) and are therefore accepted as indicativeof myoprotective activity. Exemplary stressor assays for this purposeare provided in the Examples. For example, cardiomyocytes in cultureexhibit contractile (“beating”) activity; each cardiomyocyte contractionis associated with a rise in intracellular calcium termed “calcium”.These calcium transients can be measured using Fluo-4, a fluorescent dyewhich exhibits large fluorescence intensity increases upon the bindingof calcium. This assay is cell-based and tests the ability of potentialcytoprotectant molecules to guard against ischemic damage and allow thecells to maintain their contractile function.

Further validation of compounds can be carried out in a whole organassay, such as the isolated heart (Langendorff) model of cardiacfunction. Similarly, compounds can be further validated in additionalanimal models of disease (e.g., diabetes, renal failure, asthma, musclefatigue, inflammation), such as are well known in the art.

Further validation of neuroantiinflammatory activity of compounds can beassessed in vitro by the inhibition of IL-1.beta. release from amicroglial cell line.

Interleukin-1 (IL-1) is a proinflammatory cytokine that exists in twoseparate forms that share 30% sequence homology (alpha and beta).Constitutive expression of IL-1 is low in the brain but levels of bothforms of this cytokine increase dramatically after injury. There issubstantial evidence that IL-1 is an important mediator ofneurodegeneration induced by cerebral ischemia (Touzani O et al, JNeuroimmunol 100:203-215,1999). Both IL-1 forms are rapidly induced inexperimental models of stroke and administration of recombinant IL-1betaenhances ischemic injury (see Hill J K. et al. Brain Res 820:45-54,1999, Hillhouse E W et al. Neurosci Lett 249:177-179, 1998, Loddick S Aet al J Cereb Blood Flow Metab 16:932-940, 1996, Stroemer R P et al., JCereb Blood Flow Metab 18:833-839, 1998). Conversely, blocking IL-1actions with a receptor antagonist or a neutralizing antibody markedlyreduces neuronal death and inflammation in models of ischemic damage(see Betz A L, J Cereb Blood Flow Metab 15:547-551, 1995, Relton J K,Brain Res Bull 29:243-246, 1992, Yamasaki Y et al, Stroke 26:676-680,1995). Furthermore, mice with decreased IL-1.beta. production (caspase-1knockouts) are significantly protected from ischemic injury (Schielke GP, et al. J Cereb Blood Flow Metab 18:180-185, 1998) and IL-1.alpha. and.beta. double knockouts exhibit dramatically reduced ischemic infarctvolumes compared with wild-type mice (87% reduction in cortex) (Boutin Het al., J Neurosci 21:5528-5534, 2001).

In addition to a role in ischemic damage, IL-1 elevation has beenassociated with many neurodegenerative diseases. There is increasingevidence for a role of IL-1 in Alzheimer's Disease (AD) (Mrak R E et al.Neurobiol Aging 22(6):903-908, 2001). Elevated levels of IL-1.beta. havebeen shown to surround amyloid plaques in the disease and recent geneticstudies have indicated that a polymorphism in IL-1.alpha. is linked toan increased risk of AD (3-6 fold increase) (Griffin W S et al., JLeukoc Biol 72(2):233-238, 2002). This polymorphism has also beencorrelated with rate of cognitive decline in AD patients (Murphy G M etal., Neurology, 56(11)1595-1597, 2001). The risk of AD is increased evenfurther when the polymorphism in IL-1.alpha. is found in combinationwith another polymorphism in IL-1.beta. (see Griffin W S, supra),providing convincing evidence that these cytokines play an importantrole in the pathology of the disease.

This assay measures the release of IL-1.beta. from a mouse microglialcell line following an inflammatory challenge with LPS andinterferon-gamma. The ability of test articles to inhibit microglialcell activation and IL-1beta release is determined by co-incubation ofthe test article with the inflammatory challenge. Cytokine release ismeasured using a mouse IL-1.beta. ELISA and cell toxicity is determinedusing Cell Tracker Green (a fluorescent dye that measures cellviability).

Further in-vitro evaluation of anti-inflammatory activity can bedetermined by the well characterized E-selectin (ELAM) production assay,which measures activity of test compounds in reducing expression of ELAMin activated endothelial cells. Briefly, endothelial cells are createdby adding known activators such as lipopolysaccharides, TNF or IL-1βalone or in some combination. Activated cells produce ELAM, which can bemeasured using, for example, an E-selectin monoclonal antibody basedElisa assay. In studies carried out in support of the present invention,ELAM production was decreased as described in Example 45.

In vivo evaluation of anti-inflammatory activity can be determined bywell characterized assays measuring Carrageenan-Induced Paw Edema and byMouse Ear Inflammatory Response to Topical Arachidonic Acid. (Gabor, M.,Mouse Ear Inflammation Models and their Pharmacological Applications,2000). Carrageenan-Induced Paw Edema is a model of inflammation, whichcauses time-dependent edema formation following carrageenanadministration into the intraplantar surface of a rat paw. Theapplication of arachidonic acid (AA) to the ears of mice producesimmediate vasodilatation and erythema, followed by the abruptdevelopment of edema, which is maximal at 40 to 60 min. The onset ofedema coincides with the extravasations of protein and leukocytes. Afterone hour the edema wanes rapidly and the inflammatory cells leave thetissue so that at 6 hours the ears have returned to near normal. Theseassays, as described in Examples 42 and 43 respectively, measure a testcompound's ability to treat these inflammatory processes via systemicand topical routes of administration.

Cytoprotective activity for skin can be evaluated in cell culture usingthe Epiderm Skin Model (EPI-100) from the Mattek Corporation of Ashland,Mass. Cell cultures of neonatal foreskin are cultured in accordance withthe manufacturer's directions, and are assayed for percent cellularviability by measuring the amount of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dyetaken up by the cells, as described in Example 44.

Administration

The compounds of Formula I or III are administered at a therapeuticallyeffective dosage, e.g., a dosage sufficient to provide treatment for thedisease states previously described. Administration of the compounds ofthe invention or the pharmaceutically acceptable salts thereof can bevia any of the accepted modes of administration for agents that servesimilar utilities.

While human dosage levels have yet to be optimized for the compounds ofthe invention, generally, a daily dose is from about 0.01 to 2.0 mg/kgof body weight, preferably about 0.1 to 1.5 mg/kg of body weight, andmost preferably about 0.3 to 1.0 mg/kg of body weight. Thus, foradministration to a 70 kg person, the dosage range would be about 0.7 to140 mg per day, preferably about 7.0 to 105 mg per day, and mostpreferably about 21 to 70 mg per day. The amount of active compoundadministered will, of course, be dependent on the subject and diseasestate being treated, the severity of the affliction, the manner andschedule of administration and the judgment of the prescribingphysician.

The compositions of the present invention are suitable for providingprotection against the harmful effects of ultraviolet radiation,preferably in personal care products. More preferably, the compositionsof the present invention are suitable for use as sunscreens to provideprotection to human skin from the harmful effects of UV radiation, whichinclude, but are not limited to, sunburn and premature aging of theskin. The present invention therefore also further relates to methods ofprotecting human skin from the harmful effects of UV radiation. Suchmethods generally involve attenuating or reducing the amount of UVradiation that reaches the skin's surface. In the case of the presentinvention, the methods of treatment for the harmful effects ofultraviolet radiation also include administration of a composition ofthe invention after the exposure to UV radiation has already takenplace. To protect the skin from UV radiation, a safe and effective(photoprotective) amount of the composition is topically applied to theskin. “Topical application” refers to application of the presentcompositions by spreading, spraying, etc. onto the surface of the skin.The exact amount applied may vary depending on the level of UVprotection desired. From about 0.5 mg of composition per squarecentimeter of skin to about 25 mg of composition per square centimeterof skin are typically applied.

Compounds and methods of the invention may be employed in any skin careapplication where decreased inflammatory response is desirable. Forexample, compounds and compositions of the invention may be incorporatedinto leave-on and rinse-off acne preparations, facial milks andconditioners, shower gels, foaming and non-foaming facial cleansers,cosmetics, hand and body lotions, leave-on moisturizers, cosmetic andcleaning wipes, salves for poison ivy, chicken pox, or pruritis, or thelike. Generally, for dermal applications, topical administration ispreferred; however, systemic administration, as described elsewhereherein, is also possible.

Compositions of the present invention may also be used in cosmeticcompositions. Cosmetic compositions of the present invention are ideallysuited for use in treating the skin and lips, especially in the form ofa lipstick or lip balm for applying to the lips a permanent orsemi-permanent color, ideally with a gloss or luster finish. Thecosmetic compositions can also be used in treating the skin and/or lipswith a skin care agent for protection against exposure to adverseweather, including the wind and rain, dry and/or hot environments,environmental pollutants (e.g., ozone, smoke, and the like), or exposureto excessive doses of sunlight. The compositions are also useful inproviding sun protection, moisturizing and/or conditioning for the hairand skin, improved skin feel, regulating skin texture, reducing finelines and wrinkles, reducing oily shine on hair or skin, skin lighteningand reducing skin or hair odor. The cosmetic compositions canaccordingly be applied to the skin and/or lips in the traditional mannerwith or without a conventional holder or applicator to provide adecorative and/or protective film thereto.

In employing the compounds of this invention for treatment of the aboveconditions, any pharmaceutically acceptable mode of administration canbe used. The compounds of Formula I or III can be administered eitheralone or in combination with other pharmaceutically acceptableexcipients, including solid, semi-solid, liquid or aerosol dosage forms,such as, for example, tablets, capsules, powders, liquids, suspensions,suppositories, aerosols or the like. The compounds of Formula I can alsobe administered in sustained or controlled release dosage forms,including depot injections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.The compositions will typically include a conventional pharmaceuticalcarrier or excipient and a compound of Formula I or III or apharmaceutically acceptable salt thereof. In addition, thesecompositions may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, and the like, including, but not limited toanticoagulants, blood clot dissolvers, permeability enhancers and slowrelease formulations.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,preferably about 0.5% to 50%, by weight of a compound or salt of FormulaI or III, the remainder being suitable pharmaceutical excipients,carriers, etc.

One preferred manner of administration for the conditions detailed aboveis oral, using a convenient daily dosage regimen which can be adjustedaccording to the degree of affliction. For such oral administration, apharmaceutically acceptable, non-toxic composition is formed by theincorporation of any of the normally employed excipients, such as, forexample, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like. Such compositions take theform of solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations and the like.

Preferably the compositions will take the form of a pill or tablet andthus the composition will contain, along with the active ingredient, adiluent such as lactose, sucrose, dicalcium phosphate, or the like; alubricant such as magnesium stearate or the like; and a binder such asstarch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose andderivatives thereof, and the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier, such as, forexample, water, saline, aqueous dextrose, glycerol, glycols, ethanol,and the like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, sodium acetate, sodium citrate, cyclodextrinederivatives, sorbitan monolaurate, triethanolamine acetate,triethanolamine oleate, etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 15th Edition, 1975. The composition or formulationto be administered will, in any event, contain a quantity of the activecompound in an amount effective to alleviate the symptoms of the subjectbeing treated.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 95% with the balance made up from non-toxic carrier may beprepared.

For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, talcum, cellulosederivatives, sodium crosscarmellose, glucose, sucrose, magnesiumcarbonate, sodium saccharin, talcum and the like. Such compositions takethe form of solutions, suspensions, tablets, capsules, powders,sustained release formulations and the like. Such compositions maycontain 0.01%-95% active ingredient, preferably 0.1-50%.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is preferablyencapsulated in a gelatin capsule. Such diester solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, thesolution, e.g. in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g. water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g. propylenecarbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

Other useful formulations include those set forth in U.S. Pat. Nos. Re.28,819 and 4,358,603.

The formulation can be administered in a single unit dosage form forcontinuous treatment or in a single unit dosage form ad libitum whenrelief of symptoms is specifically required. For example, theformulation may be administered as a bolus or as a continuousintravenous infusion after onset of symptoms of stroke, myocardialinfarction or chronic heart failure.

Another preferred manner of administration is the topicaladministration. “Topical administration” refers to application of thepresent compositions by spreading, spraying, etc. onto the surface ofthe skin. The typical amount applied may vary from about 0.1 mg ofcomposition per square centimeter of skin to about 25 mg of compositionper square centimeter of skin. The compounds of the present inventionmay be formulated for topical administration to the epidermis asointments, creams or lotions or as transdermal patch. Formulationssuitable for topical administration in the mouth include lozenges,pastilles and mouthwashes.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents, solubilityenhancers, and the like, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate, cyclodextrins, etc.

A more recently devised approach for parenteral administration employsthe implantation of a slow-release or sustained-release system, suchthat a constant level of dosage is maintained. See, e.g., U.S. Pat. No.3,710,795. The percentage of active compound contained in suchparenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject. However, percentages of active ingredient of 0.01% to 10% insolution are employable, and will be higher if the composition is asolid which will be subsequently diluted to the above percentages.Preferably the composition will comprise 0.2-2% of the active agent insolution.

Nasal solutions of the active compound alone or in combination withother pharmaceutically acceptable excipients can also be administered.

Formulations of the active compound or a salt may also be administeredto the respiratory tract as an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation have diameters of less than 50 microns, preferably less than10 microns.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

General Characterization Methods

As reported in the following examples, Nuclear Magnetic Resonance (NMR)spectra were recorded on a Bruker DTX 300 spectrometer using, in mostcases, tetramethyl silane (TMS) as the internal reference. Mass spectrawere obtained on an Agilent 1100 LC/MSD instrument using eitherelectrospray ionization (positive or negative mode) (ESI) or atmosphericpressure chemical ionization (positive or negative mode) (APCI).

Example 1 Preparation of3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of 2-mercaptobenzimidaole (4.0 g) and ethyl bromopyruvate (4mL) in ethanol (40 mL) and acetone (50 mL) was stirred at 20° C. for 16hours under argon. The white solid formed was filtered to give 4.5 g ofpyruvate adduct, which was used directly for the next step withoutfurther purification. The mother liquid was concentrated and allowed toform a second crop of white solid. After filtration, it gave 4.17 g ofthe same intermediate. The first crop of the pyruvate adduct (4.5 g) wasdissolved in methylene chloride and treated with aqueous sodiumbicarbonate solution until the pH reached 7. Methylene chloride phasewas separated, dried, and concentrated to about 20 mL in volume. To thissolution was added 4-dimethylaminepyridine (700 mg) followed by stirringat 20° C. under argon. Column chromatography purification (100 g ofsilica gel, using gradient methylene chloride-ethanol mixture as theeluents) gave the title furanone product (500 mg), along with a mixtureof the product and 4-dimethylaminepyridine (2.78 g). ¹H-NMR (300 MHz,d₆-DMSO/d₆-acetone) δ (ppm) 7.50-7.45 (m, 2H), 7.40-7.35 (m, 2H),7.25-7.15 (m, 2H), 7.10-7.00 (m, 2H), 4.37 (s, 2H), 4.01 (q, J=7.1 Hz,2H), 1.13 (t, J=7.1 Hz, 3H). ¹³C-NMR (75 MHz, d₆-DMSO) δ (ppm) 166.8,266.4, 149.9, 148.9, 146.4, 139.3, 122.6, 122.0, 114.7, 112.4, 85.2,63.0, 13.9. MS (API-ESI) m/z 242, 483 (M+H⁺).

Example 2 Preparation of3-(3-Amino-[1,2,4]thiadiazol-5-ylsulfanyl)-2-(3-amino-[1,2,4]thiadiazol-5-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

2A. Preparation of the Trimethylamine Salt.

A suspension of 5-amino-1,3,4-thiadiazole-2-thiol (665 mg, 5 mmol) andethyl bromopyruvate (0.65 mL, 5.16 mmol) in ethanol (15 mL) was stirredat 20° C. under argon for 1 hour. To this solution was added atrimethylamine water solution (2 mL) to adjust pH to neutral, and it wasthen allowed to stir for additional 16 hours. A trimethylamine salt ofthe title furanone product (820 mg) was isolated after filtration.¹H-NMR (300 MHz, d₃-MeOD/D₂O) δ (ppm) 4.05-3.70 (m, 4H), 2.91 (s, 9H),1.15 (t, J=7.2 Hz, 3H). MS (API-ESI) m/z 225 ((M+2H)/2, 15), 449 (M+H⁺).

2B. Preparation of the Salt-free Furanone.

A suspension of 5-amino-1,3,4-thiadiazole-2-thiol (665 mg, 5 mmol) andethyl bromopyruvate (0.65 mL, 5.16 mmol) in ethanol (15 mL) was stirredat 20° C. under argon for 1 hour. The pH of the solution was adjusted toabout 7.5 using sodium bicarbonate aqueous solution. After 16 hoursstirring at 20° C., the precipitate that formed was separated byfiltration. Upon drying, the title salt-free furanone product wasobtained as a solid (590 mg). ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.05-3.80(m, 4H), 1.09 (t, J=7.2 Hz, 3H). MS (API-ESI) m/z 225, 449 (M+H⁺).

Example 3 Preparation of3-(5-Amino-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-amino-2H-[1,2,4]triazol-3ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A suspension of 3-amino-5-mercapto-1,2,4-triazole (580 mg, 5 mmol) andethyl bromopyruvate (0.65 mL, 5.16 mmol) in ethanol (15 mL) was stirredat 20° C. under argon for 2 hours. The pH of the solution was adjustedto about 7.5 using sodium bicarbonate aqueous solution. Afterevaporation to dryness, the residue was extracted with hot ethanol. Uponcooling, the ethanol solution was filtered and the filtrate wasevaporated to dryness, giving the title furanone product (650 mg) as alight brown powder. ¹H-NMR (300 MHz, D₂O) δ (ppM) 4.00-3.60 (m, 4H),1.01 (t, J=7.2 Hz, 3H). MS (API-ESI) m/z 208, 415 (M+H⁺).

Example 4 Preparation of4-Hydroxy-3-(5-nitro-1H-benzoimidazol-2-ylsulfanyl)-2-(5-nitro-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of 3-mercapto-5-nitrobenzimidazole (975 mg, 5 mmol) and ethylbromopyruvate (0.65 mL, 5.16 mmol) in acetone (10 mL) and ethanol (12mL) was stirred at 20° C. under argon for 3 hours. The pH of thesolution was adjusted to about 7.2 using sodium bicarbonate aqueoussolution. After evaporation to dryness, the residue was dissolved inethyl acetate (150 mL) and extracted with water (2×10 mL). The secondportion of the water phase (10 mL) was freeze-dried, which gave thetitle furanone product (646 mg) as a brown powder. ¹H-NMR (300 MHz,d₆-acetone) δ (ppm) 8.12 (s, 2H), 7.90-7.80 (m, 2H), 7.45-7.30 (m, 2H),4.42 (d, J=14.4 Hz, 1H), 4.25-4.10 (m, 3H), 1.11 (t, J=7.2 Hz, 3H). MS(API-ESI) m/z 573 (M+H⁺).

Example 5 Preparation of4-Hydroxy-5-oxo-3-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanyl)-2-(5-phenyl-[1,3,4]oxadiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of 5-phenyl-1,3,4-oxadiazole-2-thiol (891 mg, 5.0 mmol) andethyl bromopyruvate (0.65 mL, 5.16 mmol) in acetonitrile (100 mL) wasstirred at 20° C. under argon for 3 hours. Diisopropyl ethylamine (0.5mL) was added to the solution and it was then allowed to stir for 3additional hours. After the removal of solvents, the residue wasdissolved in ethanol (5 mL). The pH of the solution was subsequentlyadjusted to about 7.2 using sodium bicarbonate aqueous solution. Afterevaporation to dryness, the residue was dissolved in ethyl acetate (150mL) and extracted with water (2×10 mL). The second portion of the waterphase (10 mL) was freeze-dried. Further column chromatographypurification (silica gel, methylene chloride-ethanol from 95:5 to 80:20)gave the title furanone product (80 mg). ¹H-NMR (300 MHz, CDCl₃) δ (ppm)7.84 (d, J=7.3 Hz, 1H), 7.75 (d, J=7.3 Hz, 1H), 7.50-7.20 (m, 6H), 4.07(d, J=14.1 Hz, 1H), 4.00-3.85 (m, 3H), 1.02 (t, J=7.1 Hz, 3H). MS(API-ESI) m/z 539 (m+H⁺).

Example 6 Preparation of3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of 5-chloro-2-mercaptobenzothiazole (1.01 g, 5 mmol) andethyl bromopyruvate (0.65 mL, 5.16 mmol) in acetonitrile (100 mL) wasstirred at 20° C. under argon for 3 hours. The precipitates werefiltered off and washed with acetonitrile. Drying under vacuum, affordedthe corresponding pyruvate adduct (1.68 g) as a solid. A portion of thisintermediate (200 mg) was dissolved in ethanol (5 mL). The pH of thesolution was adjusted to about 7.2 using sodium bicarbonate aqueoussolution. After evaporation to dryness, the residue was purified usingcolumn chromatography method (silica gel, methylene chloride-ethanolfrom 95:5 to 80:20) gave the title furanone product (72 mg). ¹H-NMR (300MHz, CDCl₃) δ (ppm) 7.84 (d, J=8.3 Hz, 1H), 7.78 (d, J=2.0 Hz, 1H), 7.76(d, J=8.5 Hz, 1H), 7.70 (d, J=2.1 Hz, 1H), 7.30 (dd, J=2.1 Hz, J=8.5 Hz,1H), 7.21 (dd, J=2.1 Hz, 8.5 Hz, 1H), 4.63 (d, J=14.1 Hz, 1H), 4.21 (d,J=14.1 Hz, 1H), 4.10-3.85 (m, 2H), 1.00 (t, J=7.1 Hz, 3H) ppm. MS(API-ESI) m/z 233 (100), 585 (M+H⁺).

Example 7 Preparation of4-Hydroxy-3-(5-methoxy-1H-benzoimidazol-2-ylsulfanyl)-2-(5-methoxy-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of 5-methoxy-2-benzimidazole thiol (901 mg, 5 mmol) and ethylbromopyruvate (0.65 mL, 5.16 mmol) in ethanol (10 mL) and acetone (20mL) was stirred at 20° C. under argon for 3 hours. The precipitates werefiltered off and washed with ethyl acetate. Drying under vacuum affordedthe corresponding pyruvate adduct intermediate (1.20 g) as a solid. Aportion of the intermediate (300 mg) was dissolved in ethanol (5 mL).The pH of the solution was adjusted to about 7.2 using sodiumbicarbonate aqueous solution. After evaporation to dryness, the residuewas dissolved in ethyl acetate and washed with water. After drying overanhydrous magnesium sulfate, the organic phase was evaporated todryness, to give the title furanone product (240 mg). ¹H-NMR (300 MHz,d₆-DMSO/d₆-acetone) δ (ppm) 7.37 (d, J=8.8 Hz, 1H), 7.29 (d, J=8.7 Hz,1H), 6.98 (d, J=2.4 Hz, 1H), 6.89 (d, J=2.4 Hz, 1H), 6.81 (dd, J=2.4 Hz,J=8.8 Hz, 1H), 6.69 (dd, J=2.4 Hz, J=8.7 Hz, 1H), 4.31 (d, J=14.3 Hz,1H), 4.25 (d, J=14.3 Hz, 1H), 4.10-4.00 (m, 2H), 3.79 (s, 3H), 3.77 (s,3H), 1.09 (t, J=7.1 Hz, 3H). MS (API-ESI) m/z 272, 543 (M+H⁺).

Example 8 Preparation of4-Hydroxy-5-oxo-3-p-tolylsulfanyl-2-p-tolylsulfanylmethyl-2,5-dihydro-furan-2-carboxylicacid ethyl ester

To a solution of 4-methyl benzenethiol (248 mg, 2 mmol) in DCM (10 mL)was added ethyl bromopyruvate (431 mg, 2.0 mmol, in 2 mL AcCN) dropwisewith vigorous stirring. Upon completion of the addition, the reactionwas stirred for another 30 min. The mixture was then washed with water(2×5 mL) and dried over Na₂SO₄. After solvent evaporation, the residuewas chromatographed to afford a pyruvate adduct intermediate (1:1mixture of enol and keto form) as a pale yellow oil (310 mg, 65%).¹H-NMR (300 MHz, CD₃OD) δ (ppm) 7.37-7.27 (m, 2H), 7.17-7.09 (m, 2H),6.55 (s, 0.22H), 4.37-4.20 (m, 2H), 3.89 (s, 0.72H), 2.34-2.30 (m, 3H),1.44-1.26 (m, 3H). MS-ESI (2M+H⁺) m/z 477.

To the solution of the intermediate (102 mg, 0.43 mmol) in DCM (5 mL)was added 3 drops of TEA (in 0.2 mL of DCM). The reaction was monitoredusing MS. Upon the completion of the reaction (3 h), the reactionmixture was diluted with EtOAc (30 mL) and washed with aqueous HClsolution (0.1M, 10 mL). After solvent evaporation, the residue waschromatographed to give the title furanone product as a pale yellow oil(53 mg, 58%). ¹H-NMR (300 MHz, CD₃OD) δ (ppm) 7.33-7.29 (m, 4H),7.11-7.08 (m, 2H), 4.40 (s, 1H), 4.05-3.98 (m, 2H), 3.70-3.58 (m, 2H),2.32 (s, 3H), 2.31 (s, 3H), 1.17 (t, J=7.2 Hz, 3H). ¹³CNMR (75 MHz) δ(ppm) 167.5, 166.9, 138.8, 137.5, 132.3, 132.1, 131.6, 130.0, 129.9,129.8, 125.7, 86.6, 62.9, 41.0, 21.2, 21.1, 13.8.

Example 9 Preparation of3-(2-Acetylamino-2-methoxycarbonyl-ethylsulfanyl)-2-(2-acetylamino-2-methoxycarbonyl-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

To a solution of 2-acetylamino-3-mercapto-propionic acid methyl ester(177 mg, 1 mmol) in acetonitrile (5 mL) at room temperature withstirring was added ethyl bromopyruvate (215 mg, 1.0 mmol, in 1.0 mLAcCN) dropwise. The reaction was monitored using MS. Upon completion,the reaction was quenched by adding 30 mL of EtOAc. After solventevaporation, the residue was chromatographed using DCM/MeOH (8:1) as theeluents to afford the corresponding pyruvate adduct as a yellow oilcontaining both enol and keto forms (151 mg, 52%). ¹H-NMR (300 MHz,CD₃OD) δ (ppm) 6.18 (s, 0.2H), 4.85-4.72 (m, 1H), 4.33-4.19 (m, 2H),3.71 (s, 3H), 3.59-3.57 (m, 0.8H), 3.25-2.81 (m, 2H), 2.02-1.98 (m, 3H),1.35-1.23 (m, 3H). ¹³C-NMR (75 MHz) δ (ppm) 185.9, 171.0, 170.3, 170.2,130.1, 162.6, 160.7, 138.0, 111.6, 62.9, 62.1, 52.7, 52.5, 51.7, 51.5,40.5, 37.1, 35.7, 34.1, 22.9, 14.2, 13.9. MS-ESI (M+H⁺) m/z 292.

To a solution of the pyruvate adduct (85 mg) in AcCN (3 mL) was added 5drops of Na₂CO₃ (2.0 M). The resulting suspension was stirred for 3 huntil MS indicated the completion of the condensation. The reaction wasquenched by adding EtOAc (20 mL) and the mixture was then dried overNa₂SO₄. After solvent evaporation, the residue was chromatographed(DCM/MeOH, 6:1) to give the title furanone product as a pale yellow oil(42 mg, 53%). ¹H-NMR (300 MHz, CD₃OD) δ (ppm) 4.86-4.75 (m, 2H),4.29-4.21 (m, 2H), 3.76 (s, 3H), 3.75 (s, 3H), 3.65-3.06 (m, 6H), 2.07(s, 3H), 2.04 (s, 3H), 1.32-1.24 (m, 3H). ¹³C-NMR (75 MHz) δ (ppm)171.4, 171.3, 171.0, 170.7, 167.6, 147.3, 128.4, 87.0, 63.1, 53.0,52.84, 52.75, 52.8, 52.1, 37.7, 35.7, 34.9, 33.7, 22.97, 22.91, 13.98.MS-ESI (M+H⁺) m/z 537.

Example 10 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a TFA salt of the Diastereoisomer Mixture

To a solution of 1-glutathione(2-amino-4-[1-(carboxymethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-butyricacid) (8.14 g, 26.5 mmol) in water (50 mL) and methanol (10 mL)(degassed and purged with nitrogen) was added bromo-3-ethylpyruvate(5.17 g, 26.5 mmol) at room temperature. Upon the addition ofbromo-3-ethylpyruvate, the cloudy suspension turned to yellowishtranslucent almost instantly. After stirring for 2 hours at roomtemperature, the mixture was concentrated on a rotary evaporator underthe reduced pressure. The solution was then washed with methylenechloride thoroughly. The organic layer was discarded. The aqueous layerwas evaporated to dryness under reduced pressure. After freeze-dryingunder high vacuum for 48 hours, a yellowish solid was obtained as thepyruvate-glutathione adduct (13.0 g, 98.0%). NMR data indicate thatthere exist two tautomeric forms of the product, namely the keto form,2-amino-4-[1-(carboxymethyl-carbamoyl)-2-(2-carboxy-2-oxo-ethylsulfanyl)-ethylcarbamoyl]-butyricacid ethyl ester, and the enol form,3-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-hydroxy-acrylicacid ethyl ester. ¹H-NMR (D₃COD, 300 MHz) δ (ppm): 1.17 (t, 3H), 1.93(m, 2H), 2.11 (m, 2H), 2.75-3.28 (m, 2H), 2.29 (ss, 1.6H), 3.93 (s, 2H),4.09 (m, 1H), 4.25 (q, 2H), 4.83 (m, 1H), 6.43 (s, 0.4H). ¹³C-NMR(D₃COD, 75 MHz) δ (ppm): 14.1, 26.6, 32.1, 35.6, 40.0, 41.4, 53.2, 54.6,62.1, 62.7, 100.0, 113.4, 139.7, 163.4, 170.8, 172.3, and 174.1. MS(ESI) m/z: 422 (M+H⁺).

The pyruvate-glutathione adduct (1.0 g, 1.99 mmol) was dissolved inwater (10 mL) and the pH of the solution was adjusted to 7.4 using 1.0 Maq. sodium hydroxide solution. After the disappearance of the startingreactant (monitored by LC/MS), the mixture was adjusted to pH 3.0 using10% TFA in water. The solution was passed through Bio gel P2 columnusing water as the eluent to separate the product from sodium salts ofHBr and TFA. The fractions containing the product were pooled,concentrated under reduced pressure at 30° C. and finally lyophilized toobtain 0.8 g of the product as a pale yellow solid, which was found tobe about 85% pure by LC. This crude product was further purified usingreverse phase medium pressure liquid chromatography (MPLC) using waterand acetonitrile as the gradient (5 to 15% CH₃CN, 50 min; 15% AcCN,50-70 min.). The fractions containing the desired furanone were pooled,concentrated under reduced pressure at 30° C. and then lyophilized toobtain the title product as a white solid (0.7 g, 77%). ¹H-NMR (300 MHz,D₂O) δ (ppm) 4.62-4.50 (m, 2H), 4.31-4.22 (m, 2H), 4.09-4.03 (m, 2H),3.98 (brs, 4H), 3.82-3.68 (m, 1H), 3.48-3.42 (m, 1H), 3.33-2.87 (m, 4H),2.58-2.52 (m, 4H), 2.26-2.16 (m, 4H), 1.25 (m, 3H). ¹⁹F-NMR (282 MHz, noreference) δ (ppm) −76.1. MS (ESI) m/z: 797(M+H⁺, 40), 399 (M+2H⁺, 100).

Example 11 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a Salt-Free Diastereoisomer Mixture

The TFA salt of diastereoisomer mixture prepared in Example 10 (1 g,0.976 mmol) was dissolved in water (5 mL) to which was added 2equivalents of aq. 1 N NaOH (1.95 mL, 1.95 mmol) at ambient temperatureand the solution was agitated for 10 minutes. The mixture was thenpurified by reverse phase HPLC using water and acetonitrile as theeluent (5 to 15% CH₃CN, 50 min; 15% CH₃CN, 50-70 min.). The fractionscontaining the desired furanone were pooled, concentrated under vacuumat 30° C. and finally freeze-dried to obtain the title, salt-freeproduct as a white solid. ¹⁹F NMR indicated that substantially all(>99.5%, based on the relative peak intensity before and afterpurification) of the original TFA salt has been converted to salt-freeform. ¹HNMR (300 MHz, D₂O) δ (ppm) 4.61-4.51 (m, 2H), 4.32-4.23 (m, 2H),3.93 (brs, 4H), 3.82-3.63 (m, 3H), 3.48-3.41 (dd, J=5.3 Hz, 1H),3.32-3.05 (m, 3H), 2.97-2.88 (m, 1H), 2.57-2.45 (m, 4H), 2.18-2.11 (m,4H), 1.26 (m, 3H). MS-ESI (M+H⁺) m/z 797.

Example 12 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, an HCl Salt of the Diastereoisomer Mixture

To a solution of the TFA salt of the diastereoisomer mixture prepared inExample 10 (60 mg, 0.0586 mmol) in deionized water (10 mL) at 0° C. (icebath) was added dropwise a HCl solution (2.15 mL, 0.129 mmol, 0.06 M,2.2 eq). The mixture was stirred for 20 min at 0° C. and thenfreeze-dried to afford a white sticky solid (48.2 mg). ¹⁹F NMR indicatedthat substantially all (>99%, based on the relative peak intensitybefore and after HCl treatment) of the original TFA salt has beenconverted to the HCl salt. ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.50-4.38 (m,2H), 4.19-4.11 (m, 2H), 3.98-3.91 (m, 2H), 3.86-3.85 (m, 4H), 3.71-3.55(m, 1H), 3.33 (dd, J=15.0, 5.0 Hz, 1H), 3.20-2.80 (m, 4H), 2.48-2.41 (m,4H), 2.14-2.05 (m, 4H), 1.16-1.11 (m, 3H). MS-ESI (M+H⁺) m/z 797.

Example 13 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, an HBr Salt of the Diastereoisomer Mixture

The HBr salt of the diastereoisomer mixture was prepared as a paleyellow sticky solid, substituting HBr for HCl in the same proceduredescribed in Example 12. ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.48-4.36 (m,2H), 4.17-4.09 (m, 2H), 3.98-3.92 (m, 2H), 3.84 (brs, 4H), 3.68-3.55 (m,1H), 3.32 (dd, J=15.0, 5.0 Hz, 1H), 3.19-2.2.76 (m, 4H), 2.44-2.40 (m,4H), 2.12-2.03 (m, 4H), 1.16-1.12 (m, 3H). MS-ESI (M+H⁺) m/z 797.

Example 14 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a MsOH Salt of the Diastereoisomer Mixture

To a solution of salt-free diastereoisomer mixture prepared in Example11 (60 mg, 0.0753 mmol) in deionized water (10 mL) at 0° C. (ice bath)was added, dropwise, a 0.1 M aqueous methyl sulfonic acid solution (1.51mL, 0.151 mmol, 2 eq). The mixture was stirred for 20 min at 0° C. andthen freeze-dried to afford the title furanone salt as a fluffy whitesolid (61.5 mg). ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.48-4.38 (m, 2H),4.19-4.13 (m, 2H), 3.80-3.79 (m, 4H), 3.68-3.51 (m, 3H), 3.37-3.30 (m,1H), 3.23-2.80 (m, 5H), 2.67-2.66 (m, 6H), 2.43-2.37 (m, 4H), 2.06-1.98(m, 4H), 1.15 (m, 3H).

Example 15 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a TsOH Salt of the Diastereoisomer Mixture

The TsOH salt of the diastereoisomer mixture was prepared according tothe procedure described in Example 14 by treating the salt-free mixturewith 2 eq of dilute aq. TsOH. The title furanone salt was obtained as awhite fluffy solid. ¹H-NMR (300 MHz, D₂O) δ (ppm) 7.55 (d, J=7.0 Hz,4H), 7.22 (d, J=7.0 Hz, 4H), 4.48-4.38 (m, 2H), 4.19-4.12 (m, 2H),3.81-3.80 (m, 4H), 3.70-3.51 (m, 3H), 3.37-2.76 (m, 6H), 2.43-2.36 (m,4H), 2.25 (brs, 6H), 2.06-1.99 (m, 4H), 1.15 (m, 3H).

Example 16 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, an AcOH Salt of the Diastereoisomer Mixture

The acetic acid salt of the diastereoisomer mixture was preparedaccording to the procedure described in Example 14 by treating thesalt-free mixture with 2 eq of dilute aq. AcOH. The title furanone saltwas obtained as a white fluffy solid. ¹H-NMR (300 MHz, D₂O) δ (ppm)4.47-4.37 (m, 2H), 4.19-4.13 (m, 2H), 3.83-3.82 (m, 4H), 3.71-3.53 (m,3H), 3.37-2.76 (m, 6H), 2.43-2.36 (m, 4H), 2.20-2.00 (m, 4H), 1.94-1.93(m, 6H), 1.15 (m, 3H).

Example 17 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethy]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a Bis-triethylamine Salt of the Diastereoisomer Mixture

The triethylamine salt of the diastereoisomer mixture was preparedaccording to the procedure described in Example 14 by the treatment ofthe salt-free mixture with 2 eq. of dilute aq. TEA. The title furanonesalt was obtained as a white solid. ¹H-NMR (300 MHz, D₂O) δ (ppm)4.46-4.36 (m, 2H), 4.20-4.11 (m, 2H), 3.69-3.63 (m, 6H), 3.46-3.14 (m,3H), 3.06 (q, J=7.3 Hz, 12H), 3.14-2.74 (m, 3H), 2.42-2.37 (m, 4H),2.06-2.01 (m, 4H), 1.16-1.11 (m, 21H).

Example 18 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a Tetra-triethylamine Salt of the Diastereoisomer Mixture

The triethylamine salt of the diatereoisomer mixture was preparedaccording to the procedure described in Example 14 by the treatment ofthe salt-free mixture with 4 eq. of dilute aq. TEA. The title furanonesalt was obtained as a white solid. ¹H-NMR (300 MHz, D₂O) δ (ppm)4.56-4.38 (m, 2H), 4.26-4.17 (m, 2H), 3.73-3.71 (m, 4H), 3.65-3.58 (m,2H), 3.44-3.23 (m, 2H), 3.15 (q, J=7.3 Hz, 24H), 3.19-2.83 (m, 4H),2.50-2.41 (m, 4H), 2.31-2.00 (m, 4H), 1.26-1.21 (m, 39H).

Example 19 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid, a Sodium Salt of the Diastereoisomer Mixture

The sodium salt of the diatereoisomer mixture was prepared according tothe procedure described in Example 14 by the treatment of the salt freetwin mixture with 2 eq of dilute NaOH solution (0.05M). The titlefuranone salt was obtained as a white solid. ¹H-NMR (300 MHz, CD₃OD) δ(ppm) 4.48-4.36 (m, 2H), 4.22-4.10 (m, 2H), 3.72-3.59 (m, 6H), 3.36-2.97(m, 5H), 2.84-2.75 (m, 1H), 2.46-2.33 (m, 4H), 2.07-1.99 (m, 4H),1.18-1.12 (m, 3H).

Example 20 Separation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid Diasteroisomers, a TFA Salt

The two single diastereoisomers were separated from the mixture whosepreparation was described in Example 10. The separation was achieved bypreparative HPLC using water and acetonitrile gradient elution, eachcontaining 0.1% TFA (2 to 18% AcCN for 25 min and then 18% AcCN for25-30 min).

For isomer #1: ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.49 (dd, J=8.5, 5.1 Hz,2H), 4.41 (dd, J=8.5, 5.1 Hz, 2H), 4.19-4.11 (m, 2H), 3.95-3.89 (m, 2H),3.86 (brs, 4H), 3.60 (dd, J=14.2, 5.2 Hz, 1H), 3.35-3.14 (m, 3H),3.04-2.97 (dd, J=14.2, 5.2 Hz, 1H), 2.83-2.76 (dd, J=14.1, 8.7 Hz, 1H),2.48-2.41 (m, 4H), 2.13-2.05 (m, 4H), 1.14 (t, J=7.2 Hz, 3H). ¹³C-NMR(75 MHz, D₂O) δ (ppm) 174.6, 174.4, 173.15, 173.13, 172.8, 172.2, 172.1,172.05, 168.5, 167.8, 142.2, 125.9, 87.2, 64.7, 53.9, 53.5, 52.6, 41.4,37.2, 34.8, 31.9, 31.3, 31.2, 25.8, 13.5. MS-ESI m/z 797 (M+H⁺, 40), 399(M+2H⁺, 100). ¹⁹F NMR (282 MHz, no reference) δ −76.2.

For isomer #2: ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.56-4.46 (m, 2H),4.30-4.19 (m, 2H), 3.98-3.70 (m, 8H), 3.48-3.40 (m, 1H), 3.27-2.80 (m,4H), 2.55-2.40 (m, 4H), 2.20-2.10 (m, 4H), 1.25 (t, J=6.9 Hz, 3H).¹³C-NMR (75 MHz, D₂O) δ (ppm) 174.5, 173.1, 173.0, 172.6, 172.2, 171.9,171.8, 168. 167.6, 142.2, 125.8, 87.3, 64.7, 53.7, 53.4, 52.6, 41.3,37.6, 34.8, 32.0, 31.3, 31.2, 25.8, 25.7, 13.4. MS-ESI m/z 797(M+H⁺,40), 399 (M+2H⁺, 100).

Example 21 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid Diasteroisomer, an HBr Salt

By following the procedures described in Examples 13 and 20, the HBrsalts of both isomer #1 and isomer #2_were prepared (as yellow stickysolids).

For isomer #1: ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.46 (dd, J=8.5, 5.1 Hz,2H), 4.39 (dd, J=8.5, 5.1 Hz, 2H), 4.16-4.08 (m, 2H), 3.98-3.92 (m, 2H),3.85 (brs, 4H), 3.58 (dd, J=14.2, 5.2 Hz, 1H), 3.34-3.11 (m, 3H),3.01-2.95 (dd, J=14.2, 5.2 Hz, 1H), 2.81-2.73 (dd, J=14.2, 8.7 Hz, 1H),2.45=2.40 (m, 4H), 2.10-2.05 (m, 4H), 1.14 (t, J=7.1 Hz, 3H). MS-ESI(M+H⁺) m/z 797.

For isomer #2: ¹HNMR (300 MHz, D₂O) δ (ppm) 4.46-4.39 (m, 2H), 4.19-4.12(m, 2H), 4.16-4.08 (m, 2H), 3.98-3.93 (m, 2H), 3.86-3.85 (m, 4H), 3.67(dd, J=14.2, 4.2 Hz, 1H), 3.37-2.80 (m, 5H), 2.48-2.42 (m, 4H),2.14-2.04 (m, 4H), 1.14 (t, J=7.1 Hz, 3H). MS-ESI (M+H⁺) m/z 797.

Example 22 Preparation of 3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid Diasteroisomer, an HCl Salt

By following the procedures described in Examples 12 and 20, the HClsalts of both isomer #1 and isomer #2_were prepared (as white stickysolids).

For isomer #1: ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.48 (dd, J=8.5, 5.1 Hz,2H), 4.40 (dd, J=8.5, 5.1 Hz, 2H), 4.18-4.09 (m, 2H), 3.96-3.90 (m, 2H),3.86 (brs, 4H), 3.58 (dd, J=14.2, 5.2 Hz, 1H), 3.35-3.13 (m, 3H),3.03-2.96 (dd, J=14.2, 5.2 Hz, 1H), 2.82-2.75 (dd, J=14.1, 8.7 Hz, 1H),2.47-2.41 (m, 4H), 2.13-2.04 (m, 4H), 1.13 (t, J=7.2 Hz, 3H). MS-ESI(M+H⁺) m/z 797.

For isomer #2: ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.46-4.39 (m, 2H),4.20-4.13 (m, 2H), 3.95-3.90 (m, 4H), 3.86 (pseudo-d, 2H), 3.67 (dd,J=14.2, 4.2 Hz, 1H), 3.37-3.05 (m, 3H), 2.98-2.92 (dd, J=14.2, 5.2 Hz,1H). 2.87-2.80 (dd, J=14.1, 8.7 Hz, 1H), 2.47-2.39 (m, 4H), 2.13-2.04(m, 4H), 1.14 (t, J=7.1 Hz, 3H). MS-ESI (M+H⁺) m/z 797.

Example 23 Preparation of3-[3-(2-Carboxy-pyrrolidin-1-yl)-2-methyl-3-oxo-propylsulfanyl]-2-[3-(2-carboxy-pyrrolidin-1-yl)-2-methyl-3-oxo-propylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of captopril (293 mg, 1.35 mmol) and ethyl bromopyruvate(0.176 mL, 1.40 mmol) in water (1 mL) and acetonitrile (1 mL) wasstirred at 20° C. for 16 hours under argon. The pH of the solution wasadjusted to about 7.2 using sodium bicarbonate aqueous solution andstirred at 20° C. for additional 3 hours. The pH of the solution wasthen adjusted to about 4 using hydrogen bromide aqueous solution (48%).The solution was washed with methylene chloride three times. The organicphase was discarded and aqueous phase was freeze-dried. The residue wasdissolved in a solution of methylene chloride-methanol (90:10). Theclear solution was collected after centrifugation. Evaporation gave thetitle furanone product (295 mg). ¹H-NMR (300 MHz, D₂O) δ (ppm) 4.35-4.20(m, 4H), 3.75-2.70 (m, 12H), 2.25-1.85 (m, 8H), 1.30-1.05 (m, 9H). MS(API-ESI) m/z 617 (M+H⁺, 31), 639 (M+Na⁺, 100).

Example 24 Preparation of4-Hydroxy-5-oxo-3-(pyridin-4-ylsulfanyl)-2-(pyridin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester

A solution of 4-mercaptopyridine (222 mg, 2.0 mmol) and ethylbromopyruvate (0.264 mL, 2.1 mmol) in ethanol (5 mL) was stirred at 20°C. for 3 hours under argon. The pH of the solution was adjusted to about7.2 using sodium bicarbonate aqueous solution. After evaporation todryness, the residue was dissolved in ethyl acetate and washed withsodium bicarbonate aqueous solution, water, and dried over magnesiumsulfate. Evaporation and chromatography (silica gel, methylenechloride-methanol from 90:10 to 80:20) gave the title furanone product(240 mg). ¹H-NMR (300 MHz, D₃COD) δ (ppm) 8.35-8.25 (m, 2H), 8.25-8.20(m, 2H), 7.40-7.35 (m, 2H), 7.25-7.15 (m, 2H), 4.00-3.80 (m, 3H), 3.70(d, J=14.5 Hz, 1H), 1.02 (t, J=7.1 Hz, 3H). MS (API-ESI) m/z 203, 405(M+H⁺).

Example 25 Preparation of5,8-Dichloro-3-hydroxy-2-oxo-2H-1-oxo-4,9-dithia-benzo[f]azulene-10a-carboxylicacid ethyl ester

A solution of 3,6-dichloro-1,2-benzenedithiol (211 mg, 1.0 mmol), ethylbromopyruvate (0.528 mL, 4.2 mmol) and triethylamine (0.1 mL) inacetonitrile (2 mL) and acetone (4 mL) was stirred at 20° C. for 16hours under argon. After evaporation to dryness, the residue wasdissolved in ethyl acetate, washed with diluted aqueous HCl solution anddried over anhydrous magnesium sulfate. Evaporation and multiplechromatographies using two different eluate systems [(silica gel,methylene chloride-methanol from 99:1 to 98:2) and (silica gel,hexane-ethyl acetate 4:1)] in sequence repeatedly gave the titlefuranone product (20 mg). ¹H-NMR (300 MHz, D₃COD) δ (ppm) 7.33 (s, 2H),4.30-4.05 (m, 3H), 2.88 (d, J=14.3 Hz, 1H), 1.29 (t, J=7.1 Hz, 3H). MS(API-ESI) m/z 393 (M+H⁺, 100).

Example 26 Preparation of3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid

3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester (32 g) prepared as described in Example 1 was suspendedin water (400 mL) and ethanol (300 mL). The suspension was stirredcontinuously and aqueous sodium bicarbonate solution (23 g in 350 mL ofwater) was added dropwise during about 5 hours to maintain pH of around7.5 to 8. The resulting mixture was stirred at 20° C. for 2 days. Themilky mixture was put on a rotary evaporator to remove ethanol at atemperature below 40° C. under reduced pressure. The suspension became aclear solution soon after the evaporation started. During theevaporation process 400 mL of water were added to ensure completeremoval of ethanol. The solution was adjusted to pH8 and allowed to stirat 40° C. for 18 hours. The product was precipitated out by acidifyingthe solution to pH 5-6 using acetic acid water solution (12 mL of aceticacid in 230 mL of water). The solid was filtered, washed with water (40mL) and acetonitrile (40 mL), and dried under vacuum, giving3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid as a pale grey solid (16.5 g). ¹HNMR (300 MHz, D₂O) δ=7.10-6.70 (m,8H), 3.80 (d, 1H), 3.60 (d, 1H) ppm. ¹³CNMR (75 MHz, D₂O) δ=173.7,173.2, 160.5, 156.6, 149.8, 149.3, 137.9, 122.2, 122.0, 113.7, 113.4,104.5, 87.5, 37.4 ppm.

Example 27 Preparation of3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid (2-hydroxy-ethyl)-amide

A mixture of2,3-bis-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid (200 mg, 0.41 mmol), N-hydroxy succinimide (55 mg),dimethylaminopyridine (20 mg), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (85 mg) inmethylenechloride (20 mL) was stirred at room temperature for 2 h.Ethanolamine (0.03 mL) was added followed by diisopropylethylamine (0.1mL, and the mixture was stirred overnight at room temperature, andpoured into water. The organic layer was separated, dried, andconcentrated. The residue was purified by silica gel column eluting with10% methanol in dichloromethane to give 50 mg of3-(benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid (2-hydroxy-ethyl)-amide as a brown solid. ¹HNMR (CDCl₃—CD₃OD, 300MHz): δ: 7.80-7.60 (m, 4H), 7.38-7.20 (m, 4H), 4.20 (AB, J=7 Hz, 2H),3.55 (m, 2H), 3.20 (m, 2H), ppm. ¹³CNMR (CDCl₃—CD₃OD, 75 MHz): δ:168.20, 166.28, 152.77, 152.40, 135.26, 126.27, 124.70, 121.43, 121.18,121.00, 87.21, 60.56, 42.21, 39.61 ppm. MS: 532 (M+H⁺).

Example 28 Preparation of3-(Benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid

To a solution of glyoxylic acid monohydrate (98 mg, 1.03 mmol) in 6 mLmethylenechloride/methanol (5:1) was added triethylamine (418 μL, 3.0mmol) and stirred vigorously.3-(benzothiazol-2-ylsulfanyl)-2-oxo-propionic acid ethyl ester (281 mg,1.06 mmol) in 6 mL methylenechloride/methanol (5:1) was added dropwiseover a period of 3 hours and the reaction was stirred overnight (15 hrs)at room temperature. The reaction mixture was then neutralized withNaH₂PO₄ and washed with ethylacetate. The resulting very polar productremained in the aqueous layer and was purified by reverse-phase HPLC.The pure fractions were pooled and concentrated under reduced pressure.Upon evaporation of solvent,3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid precipitated out (70 mg) as a white solid which was filtered andwashed with water. The filtrate was collected and lyophilized to afforda pale yellow solid which was determined to be the triethylamine salt ofthe desired product. ¹H-NMR (MeOD, 300 MHz) δ (ppm): 7.92-7.87 (m, 2H),7.51-7.38 (m, 2H), 5.68 (s, 1H). MS (ESI) m/z: 310 (M+H⁺).

Example 29 Preparation of4-(Furan-2-ylmethylsulfanyl)-5-(furan-2-ylmethylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one

To a solution of 4-hydroxy-5-oxo-3-(2-furanylmethylsulfanyl)-2-[(2-furanylmethylsulfanyl)-methyl]-2,5-dihydrofuran-2-carboxylic acid ethyl ester (0.5 g,1.219 mmol) in anhydrous tetrahydrofuran (THF, 30 mL) was added a THFsolution of LiBH₄ (2 mL, 4 mmol) at 0-5° C. under argon atmosphere. Themixture was stirred overnight at room temperature and water (1 mL) wasslowly added. The mixture was stirred for 5 min and concentratedhydrochloric acid (1 mL) was added. The solution was mixed with water(150 mL) and extracted with methylene chloride (2×100 mL). The combinedorganic solution was washed with water (50 mL), dried and evaporated.The residue was purified by column chromatography, eluted with a mixedsolvent of hexane and ethyl acetate (3:1 to 2:1, v/v) affording 55.6 mgof 4-(furan-2-ylmethylsulfanyl)-5-(furan-2-ylmethylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one as a cream oil. ¹H NMR(CD₃OD, 300.16 MHz) δ (ppm): 7.45-7.42 (m, 2H), 6.35-6.22 (m, 4H),4.50-3.71 (m, 5H), 3.48 (d, J=12.2 Hz, 1H), 2.97 (d, J=14.8 Hz, 1H) and2.64 (d, J=14.8 Hz, 1H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm): 167.70,151.21, 150.45, 143.03, 142.38, 142.19, 125.61, 110.33, 110.00, 108.44,107.81, 88.79, 64.19, 34.07, 28.87 and 26.83. MS (ESI, rel. int.) m/z:369 (M+H⁺, 3), 391 (M+Na⁺, 100).

Example 30 Preparation of4-(2,2-Dimethyl-Propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic acid ethyl ester

To a solution of4-hydroxy-5-oxo-3-(2-furanylmethylsulfanyl)-2-[(2-furanylmethylsulfanyl)-methyl]-2,5-dihydrofuran-2-carboxylicacid ethyl ester (0.3 g, 0.73 mmol) in methylene chloride (25 mL) wereadded pyridine (0.19 mL, 2.36 mmol) and trimethylacetyl chloride (0.22mL, 1.77 mmol, 2.4 eq) under argon atmosphere. The mixture was gentlyrefluxed overnight under argon and then evaporated. The residue waspurified by column chromatography eluted with a mixed solvent of hexaneand ethyl acetate (4:1, v/v) affording 0.3181 g of4-(2,2-dimethyl-propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester as light yellow oil. ¹H NMR (CDCl₃, 300.16 MHz) δ(ppm): 7.40-7.37 (m, 2H), 6.35-6.25 (m, 4H), 4.35 (s, 2H), 4.33-4.22 (m,2H), 3.90 (d, J=14.7 Hz, 1H), 3.80 (d, J=14.7 Hz, 1H), 3.29 (d, J=15.1Hz, 1H), 2.97 (d, J=15.1 Hz, 1H), 1.37 (s, 9H) and 1.29 (t, J=7.1 Hz,3H). ¹³C NMR (CDCl₃, 75.48 MHz) δ (ppm): 174.35, 166.32, 163.84, 150.29,148.27, 144.72, 143.10, 142.64, 136.15, 111.00, 110.47, 109.44, 108.98,87.64, 63.40, 39.30, 36.15, 29.62, 28.02, 26.99 and 14.04. MS (ESI, rel.int.) m/z: 495 (M+H⁺, 25), 517 (M+Na⁺, 100).

Example 31 Preparation of3-(Furan-2-ylmethanesulfonyl)-2-(furan-2-ylmethanesulfonylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester

To a solution of4-hydroxy-5-oxo-3-(2-furanylmethylsulfanyl)-2-[(2-furanylmethylsulfanyl)-methyl]-2,5-dihydrofuran-2-carboxylic acid ethyl ester (0.5 g,1.219 mmol) in methylene chloride (25 mL) at −78° C. was addedm-chloroperoxybenzoic acid (1.216 g, 77% purity, 4.45 eq). The mixturewas stirred overnight and the temperature was left to increase naturallyto room temperature. The mixture was evaporated to dryness and theresidue was mixed with water (25 mL). After filtration, the aqueousfiltrate was evaporated and the residue was purified by columnchromatography eluted with ethyl acetate affording 0.1387 g solid with80% purity. This material was further purified by MPLC with a gradientmobile phase of 95% H2O/5% MeCN (0 min) to 20% H₂O/80% MeCN (210 min)giving 67 mg of3-(furan-2-ylmethanesulfonyl)-2-(furan-2-ylmethanesulfonylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester as a yellow solid. ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm):7.62-7.55 (m, 2H), 6.59-6.45 (m, 4H), 4.77 (d, J=14.9 Hz, 1H), 4.70 (d,J=14.9 Hz, 1H), 4.61 (s, 2H), 4.38-4.26 (m, 3H), 3.65 (d, J=15.5 Hz, 1H)and 1.32 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm): 165.45,150.21, 144.33, 142.31, 141.41, 112.81, 112.51, 111.03, 80.92, 63.71,54.77, 54.06, 53.06 and 12.81. MS (ESI, rel. int.) m/z: 492 (M+H₂O,100), 497 (M+Na⁺, 65).

Example 32 Preparation of Dimethylamino-acetic acid3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethylester

A solution of4-(1H-benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one(220 mg, 0.50 mmol), dimethylaminoacetyl chloride hydrochloride (174 mg,1.10 mmol) and triethylamine (0.280 mL, 2.00 mmol) in DMF (15 mL) wasstirred for 22 h. The reaction mixture was then passed through a plug ofSiO₂, which was then washed with hexane to remove excess DMF, andsubsequently with a 1:1 mixture of EtOAc and MeOH to collectdimethylamino-acetic acid3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethylester. Upon evaporation of solvent, the residue was subjected to columnchromatography (SiO₂: CH₂Cl₂:MeOH:Acetic Acid, 87:10:3 v/v/v) whichyielded an off-white solid which was dissolved in 0.5 M HCl andlyophilized (73 mg, 26%, GLI-1007817b). ¹H NMR (D₂O, 300.16 MHz) δ:7.20-7.38 (m, 8H), 4.67 (q, J=12.0 Hz, 2H), 3.9104.18 (m, 4H), 2.86-2.96(m, 6H).). ¹³C NMR (D₂O, 75.04 MHz) δ: 167.4, 165.4, 155.7, 147.2,145.3, 131.5, 130.9, 126.2, 126.1, 113.1, 112.7, 102.0, 85.2, 65.8,56.6, 43.7, 36.4. MS (ESI-Pos) m/z: 526.2 (M+H⁺).

Example 33

By following the procedures in the preceding examples and as describedin Schemes 1 and 2, the following compounds of Formula I were prepared:

-   -   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-thiazol-2-yl-5H-furan-2-one:        ¹HNMR (DMSO, 300 MHZ) δ (ppm): 7.83 (bs, 2H), 7.46-7.04 (m, 8H),        5.78 (s, I H), 4.55 (bs, 2H); MS 494.1 (M+H⁺).    -   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid; ¹HNMR (CDCl₃—CD₃OD, 300 MHz): δ: 7.80-7.60 (m, 4H),        7.38-7.20 (m, 4H), 4.50 (d, J=14.7 Hz, 1H), 4.21(d, J=14.7 Hz,        1H) ppm. ¹³CNMR (CDCl₃—CD₃OD, 75 MHz): δ: 172.09, 169.74,        169.32, 168.22, 156.33, 156.29, 153.87, 139.36, 139.22, 130.45,        130.14, 129.10, 128.65, 125.47, 125.34, 125.03, 124.89, 118.69,        89.43, 41.45 ppm. MS: 489 (M+H⁺)    -   3-(2-Chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂CO, 300.16 MHz) δ (ppm):        7.73-7.80 (m, 1H), 7.49-7.55 (m, 1H), 7.41-7.48 (m, 2H),        7.19-7.31 (m, 2H), 4.11 (q, J=7.1 Hz, 2H), 4.00 (d, J=14.8 Hz,        1H), 3.90 (d, J=14.8 Hz, 1H), 1.25 (t, J=7.1 Hz, 3H). ¹³C NMR        (CD₃)₂CO, 75.04 MHz) δ: 167.6, 164.3, 161.0, 137.5, 137.4,        135.6, 135.5, 133.8, 133.6, 130.7, 118.3, 117.9, 116.0, 115.8,        87.0, 63.7, 39.5, 14.2. MS (ESI-Pos) m/z: 507.0(M+H⁺).    -   Dimethylamino-acetic acid        3-(benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethyl        ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.85-7.80 (m, 4H),        7.36 (m, 4H), 4.81 (s, 2H), 4.35-4.10 (m, 4H), and 2.92 (s, 6H).        MS (ESI) m/z: 560 (M+H⁺, 100)    -   4-(Benzooxazol-2-ylsulfanyl)-5-(benzooxazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;        ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 3-7.50 (m, 4H), 7.32-7.27        (m, 4H) and 4.12-3.89 (m, 4H). ¹³C NMR (CD₃OD, 75.48 MHz) δ        (ppm): 164.11, 151.90, 151.37, 141.05, 140.67, 124.84, 124.74,        124.42, 124.22, 118.16, 117.90, 110.85, 110.01, 109.73, 88.09,        63.23 and 34.49. MS (ESI, rel. int.) m/z: 443 (M+H⁺).    -   4-(5-Chloro-benzothiazol-2-ylsulfanyl)-5-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;        ¹HNMR (300 MHz, Cl₃CD-CD₃OD) δ=7.77-7.55 (m, 4H), 7.30-7.20 (m,        2H), 4.14 (d, J=14.3 Hz, 1H), 4.00 (d, J=12.2 Hz, 1H), 3.94 (d,        J=12.2 Hz, 1H), 3.90 (d, J=14.3 Hz, 1H) ppm. ¹³CNMR (75 MHz,        Cl₃CD-CD₃OD) δ=168.5, 153.1, 152.9, 133.5, 132.5, 125.2, 124.8,        121.9, 121.8, 121.0, 120.9, 88.9, 63.8, 35.9 ppm. MS (API-ES)        m/z 547 (30), 543 (M+H⁺).    -   4-(Benzothiazol-2-ylsulfanyl)-5-(benzothiazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;        ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.76-7.62 (m, 4H), 7.39-7.21        (m, 4H), 4.05 (d, J=14.3 Hz, 1H), 3.92 (d, J=14.3 Hz, 1H) and        3.90 (s, 2H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm): 166.55,        152.52, 152.43, 135.28, 135.19, 126.15, 126.04, 124.59, 124.42,        121.08, 120.97, 120.63, 88.72, 63.91 and 36.01. MS (ESI, rel.        int.) m/z: 475 (M+H⁺).    -   3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂CO, 300.16 MHz) δ (ppm):        7.22-7.39 (m, 4H), 7.05-7.18 (m, 2H), 4.61 (d, J=12.4 Hz, 1H),        4.48 (d, J=12.4 Hz, 1H), 4.17 (q, J=7.1 Hz, 2H), 3.90-4.05 (m,        2H), 3.43 (d, J=14.8 Hz, 1H), 3.13 (d, J=14.8 Hz, 1H), 1.23 (t,        J=7.1 Hz, 3H). ¹³C NMR ((CD₃)₂CO, 75.04 MHz) δ: 166.9, 166.0,        163.0, 162.8, 159.7, 159.5, 144.1, 135.2, 135.0, 130.4, 130.3,        129.7, 129.6, 125.7, 124.9, 124.7, 123.9, 123.5, 114.7, 114.6,        114.4, 114.3, 62.7, 37.0, 26.8, 20.2, 13.6. MS (ESI-Pos) m/z:        535.0 (M+H⁺)    -   3-(5,6-Dichloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5,6-dichloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂SO, 300.16 MHz) δ (ppm):        7.58-7.64 (m, 4H), 4.21 (d, J=14.1 Hz, 1H), 4.14 (d, J=140.0 Hz,        1H), 3.90-4.02 (m, 2H), 0.98 (t, J=7.1 Hz, 3H). ¹³C NMR        ((CD₃)₂SO, 75.04 MHz) δ: 169.2, 168.4, 156.4, 154.6, 153.1,        124.3, 124.2, 115.6, 84.5, 62.5, 31.2, 14.1. MS (ESI-Pos) m/z:        621.0 (M+H⁺)    -   4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂CO, 300.16 MHz) δ (ppm):        7.65-7.73 (m, 2H), 7.31-7.38 (m, 2H), 6.90-6.98 (m, 2H), 4.44        (d, J=14.2 Hz, 1H), 4.20 (d, J=14.8 Hz, 1H), 3.94-4.05 (m, 2H),        3.90 (s, 3H), 3.83 (s, 3H), 1.02 (t, J=7.1 Hz, 3H). ¹³C NMR        ((CD₃)₂CO, 75.04 MHz) δ: 167.2, 166.2, 159.2, 159.1, 154.2,        127.4, 127.0, 121.6, 114.2, 104.7, 104.6, 86.6, 62.4, 55.2,        55.1, 38.3, 13.3. MS (ESI-Pos) m/z: 577.1(M+H⁺).    -   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CDCl₃, 300.16 MHz) δ (ppm): 7.09-7.31        (m, 6H), 4.27-4.50 (m, 2H), 3.98-4.21 (m, 2H), 3.65-3.83 (m,        2H), 3.18 (d, J=15.1 Hz, 1H), 2.83 (d, J=15.0 Hz, H) 1.22 (t,        J=7.0 Hz, 3H). ¹³C NMR (CDCl₃, 75.04 MHz) δ (ppm): 167.5, 166.7,        141.2, 135.0, 134.8, 134.3, 134.0, 133.8, 133.3, 132.0, 131.9,        129.7, 129.6, 127.4, 127.2, 126.9, 87.7, 63.3, 36.6, 34.9, 32.2,        14.0. MS (ESI-Pos) m/z: 569.0 (M+H⁺).    -   2-(Benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 8.17-8.06        (m, 2H), 7.91-7.83 (m, 2H), 7.64-7.57 (m, 2H), 7.47-7.38 (m,        2H), 4.28 (d, J=14.1 Hz, 1H), 4.22 (d, J=14.1 Hz, 1H), 4.08-3.95        (m, 2H) and 1.12 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ        (ppm): 175.39, 165.88, 164.63, 163.25, 157.15, 153.55, 152.96,        136.17, 135.75, 127.09, 126.54, 126.44, 125.14, 123.80, 122.37,        121.56, 121.23, 113.12, 83.15, 63.31, 59.53 and 12.67. MS (ESI,        rel. int.) m/z: 533 (M+H⁺).    -   4-(Furan-2-ylmethylsulfanyl)-5-(furan-2-ylmethylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;        ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.45-7.42 (m, 2H), 6.35-6.22        (m, 4H), 4.50-3.71 (m, 5H), 3.48 (d, J=12.2 Hz, 1H), 2.97 (d,        J=14.8 Hz, 1H) and 2.64 (d, J=14.8 Hz, 1H). ¹³C NMR (CD₃OD,        75.48 MHz) δ (ppm): 167.70, 151.21, 150.45, 143.03, 142.38,        142.19, 125.61, 110.33, 110.00, 108.44, 107.81, 88.79, 64.19,        34.07, 28.87 and 26.83. MS (ESI, rel. int.) m/z: 369 (M+H⁺, 3),        391 (M+Na⁺, 100).    -   4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂CO, 300.16 MHz) δ: 8.86 (d,        J=2.3 Hz, 1H), 8.81 (d, J=2.3 Hz, 1H), 8.24-8.28 (m, 2H), 7.98        (d, J=9.0 Hz, 1H), 7.92 (d, J=9.0 Hz, 1H), 4.80 (d, J=14.7 Hz,        1H), 4.41 (d, J=14.7 Hz, 1H), 4.23 (q, J=7.1 Hz, 2H), 1.23 (t,        J=7.1 Hz, 3H). MS (ESI-Pos) m/z: 607.0 (M+H⁺).    -   2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.72-7.66        (m, 4H), 7.57-7.53 (m, 2H), 7.49-7.37 (m, 2H), 4.53-4.29 (m,        6H), 4.0 (q, J=7.1 Hz, 2H), 1.48 (t, J=7.2 Hz, 3H), 1.18 (t,        J=7.1 Hz, 3H) and 0.97 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48        MHz) δ (ppm): 165.97, 163.47, 148.88, 147.86, 143.07, 140.42,        134.93, 133.37, 126.18, 124.93, 124.33, 120.70, 117.75, 113.48,        111.12, 85.10, 68.09, 63.94, 40.60, 37.83, 14.20, 14.07 and        13.10. MS (ESI) m/z: 539 (M+H⁺, 389).    -   3-(Furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.62-7.59        (m, 2H), 6.56-6.47 (m, 4H), 4.74 (d, J=14.2 Hz, 1H), 4.58 (d,        J=14.2 Hz, 1H), 4.44-4.26 (m, 4H), 4.09 (d, J=14.4 Hz, 1H), 3.23        (d, J=14.4 Hz, 1H) and 1.32 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD,        75.48 MHz) δ (ppm): 165.89, 163.88, 161.85, 144.19, 144.08,        144.03, 143.84, 126.15, 112.33, 111.91, 111.11, 110.89, 82.27,        63.61, 56.31, 50.87, 50.69 and 12.83. MS (ESI, rel. int.) m/z:        443 (M+H⁺), 465 (M+Na⁺, 97).    -   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.62-7.59        (m, 2H), 6.58-6.48 (m, 4H), 4.82 (d, J=14.8 Hz, 1H), 4.72 (d,        J=14.8 Hz, 1H), 4.43-4.26 (d, J=14.5 Hz, 1H), 3.16 (d, J=14.5        Hz, 1H) and 1.32 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ        (ppm): 165.74, 152.38, 149.50, 144.35, 144.09, 143.94, 141.26,        121.88, 112.98, 111.92, 111.07, 110.88, 82.19, 63.68, 55.95,        54.21, 50.74 and 12.82. MS (ESI, rel. int.) m/z: 459 (M+H⁺).    -   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester (single isomer) ¹H NMR (CD₃OD, 300.16 MHz) δ        (ppm): 7.59-7.57 (m, 2H), 6.55-6.46 (m, 4H), 4.83 (d, J=15.0 Hz,        1H), 4.70 (d, J=15.0 Hz, 1H), 4.32 (d, J=9.1 Hz, 1H), 4.30 (q,        J=7.1 Hz, 2H), 4.20 (d, J=14.2 Hz, 1H), 3.70 (d, J=14.3 Hz, 1H),        3.60 (d, J=14.3 Hz, 1H) and 1.32 (t, J=7.1 Hz, 3H). ¹³C NMR        (CD₃OD, 75.48 MHz) δ (ppm): 165.66, 150.20, 144.27, 143.96,        143.74, 141.52, 112.81, 111.85, 111.08, 110.85, 82.76, 63.64,        54.19, 54.10, 50.66 and 12.83. MS (ESI, rel. int.) m/z: 459        (M+H⁺).    -   4-Hydroxy-3-methylsulfanyl-2-methylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 4.28 (q,        J=7.2 Hz, 2H), 3.3 (d, J=14.9 Hz, 1H), 3.14 (d, J=14.9 Hz, 1H),        2.66 (s, 3H), 2.20 (s, 3H), 1.13 (t, J=7.2 Hz, 3H). ¹³C-NMR δ        (ppm): 168.1, 167.0, 139.8, 129.3, 87.8, 63.1, 40.1, 18.1, 14.3,        14.1. MS (ESI) m/z: 279 (M+H⁺).    -   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid; ¹HNMR (300 MHz, D₃COD) δ=3.40 (d, 1H), 3.60 (d, 1H) ppm.        ¹³CNMR (75 MHz, D₃COD) δ=173.3, 172.9, 172.1, 171.1, 159.0,        158.0, 155.4, 104.8, 87.5, 41.5 ppm. MS (ESI) m/z 421 (M+H⁺).    -   3-(Benzooxazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid methyl ester; ¹HNMR (300 MHz, D₃COD) δ (ppm): 7.87-7.73 (m,        4H), 7.31-7.45 (m, 4H) 4.39 (d, J=14.4 Hz, 1H), 4.29 (d, J=14.4        Hz, 1H), 3.58 (s, 3H). ¹HNMR (300 MHz, D₃COD) δ (ppm): 7.87-7.73        (m, 4H), 7.31-7.45 (m, 4H) 4.39 (d, J=14.4 Hz, 1H), 4.29 (d,        J=14.4 Hz, 1H), 3.58 (s, 3H). ¹HNMR (300 MHz, D₃COD) δ (ppm):        7.87-7.73 (m, 4H), 7.31-7.45 (m, 4H) 4.39 (d, J=14.4 Hz, 1H),        4.29 (d, J=14.4 Hz, 1H), 3.58 (s, 3H).    -   4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂CO, 300.16 MHz) δ (ppm):        7.60-7.66 (m, 6H), 7.47 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.4 Hz,        2H), 6.57 (d, J=2.7 Hz, 1H), 6.51 (d, J=2.7 Hz, 1H), 3.81-4.03        (m, 4H), 3.76 (s, 6H), 1.14 (t, J=7.1 Hz, 3H). MS (ESI-Neg) m/z:        569.1[M−H]⁻.    -   3-Hydroxy-5,6-dimethyl-2-oxo-5,6-dihydro-2H-1-oxa-4,7-dithia-azulene-8a-carboxylic        acid ethyl ester; ¹H NMR ((CD₃)₂CO, 300.16 MHz) δ (ppm): 4.23        (q, J=6.3 Hz, 2H), 3.72-3.89 (m, 1H), 2.94-3.53 (m, 3H), 1.62        (d, J=6.9 Hz, 0.4H), 1.51 (d, J=6.8 Hz, 0.4H), 1.41 (d, J=6.8        Hz, 2.6H), 1.23 (d, J=7.2 Hz, 2.6H), 1.25 (t, J=7.1 Hz, 3H). MS        (ESI-Pos) m/z: 305.1 (M+H⁺).    -   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CDCl₃, 300.16 MHz) δ (ppm): 7.39-7.36        (m, 2H), 6.35-6.25 (m, 4H), 4.35 (s, 2H), 4.33-4.24 (m, 2H),        3.90 (d, J=14.7 Hz, 1H), 3.29 (d, J=15.2 Hz, 1H), 2.97 (d,        J=15.2 Hz, 1H), 2.82 (septet, J=7.0 Hz, 1H), and 1.41-1.27 (m,        9H). ¹³C NMR (CDCl₃, 75.48 MHz) δ (ppm): 172.70, 166.28, 163.89,        150.27, 148.36, 144.90, 143.09, 142.65, 135.96, 111.01, 110.47,        109.43, 108.99, 87.65, 63.41, 36.18, 33.90, 29.62, 28.07, 18.75,        18.69 and 14.04. MS (ESI, rel. int.) m/z: 481 (M+H⁺, 25), 503        (M+Na⁺, 100).    -   4-(2,2-Dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CDCl₃, 300.16 MHz) δ (ppm) 4.40-4.30        (m, 2H), 4.27-4.19 (m, 4H), 3.96 (d, J=16.2 Hz, 1H), 3.85 (d,        J=16.2 Hz, 1H), 3.61 (d, J=15.2 Hz, 1H), 3.44 (d, J=15.3 Hz,        1H), 3.37 (d, J=15.3 Hz, 1H), 3.34 (d, J=15.2 Hz, 1H), 1.38 (s,        9H) and 1.27 (t, J=7.1 Hz, 3H). ¹³C NMR (CDCl₃, 75.48 MHz) δ        (ppm): 174.35, 170.11, 166.98, 166.18, 163.48, 143.55, 136.14,        87.18, 63.58, 62.60, 61.64, 39.31, 37.68, 34.85, 32.75, 26.94,        14.21, 14.14 and 14.06. MS (ESI) m/z: 507 (M+H⁺, 12) and 529        (M+Na⁺, 100).    -   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.84-7.82 (d,        2H), 7.69-7.66 (d, 2H), 7.42-7.28 (m, 6H), 7.19 (s, 2H)        4.49-4.11 (m, 4H), 1.78-1.23 (t, 3H). MS (ESI) m/z: 569 (M+H⁺).    -   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid; ¹H NMR (D₂O, 300.16 MHz) δ (ppm) 3.42-3.21 (m, 7H), 3.06        (d, J=14.9 Hz, 1H), 2.90-2.82 (m, 2H) and 2.76-2.74 (m, 12H).        ¹³C NMR (D₂O, 75.48 MHz) δ (ppm) 170.72, 167.76, 141.79, 125.21,        88.01, 56.95, 56.30, 42.93, 42.80, 42.77, 42.59, 27.26 and        24.75. MS (ESI) m/z=321, (M+H—CO₂, 47), 365 (M+H⁺).    -   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.39-7.33        (m, 2H), 6.33-6.30 (m, 2H), 6.24-6.22 (m, 2H), 4.47 (d, J=14.4        Hz, 1H), 4.31 (d, J=14.4 Hz, 1H), 4.29-4.19 (m, 2H), 3.84 (d,        J=14.7 Hz, 1H), 3.74 (d, J=14.7 Hz, 1H), 3.25 (d, J=15.1 Hz,        1H), 2.80 (d, J=15.1 Hz, 1H) and 1.29 (t, J=7.1 Hz, 3H). ¹³C NMR        (CD₃OD, 75.48 MHz) δ (ppm): 167.14, 166.59, 150.46, 149.63,        142.77, 142.61, 140.55, 126.29, 110.80, 110.43, 109.10, 108.73,        87.94, 63.14, 35.95, 29.63, 27.70 and 14.07. MS (ESI) m/z: 411        (M+H⁺, 5) and 433 (M+Na⁺, 100).    -   4-Hydroxy-3-(1-methyl-1H-imidazol-2-ylsulfanyl)-2-(1-methyl-1H-imidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (MeOD, 300 MHz) δ (ppm): 7.70-7.46 (4        d, 4H combined), 4.17-4.11 (m, 2.8H), 4.01 (s, 0.76H), 3.92-3.89        (d, 6H), 3.83-3.78 (2 s, 1H), 1.34-1.24 (t, 3H). MS (ESI) m/z:        411 (M+H⁺).    -   3-Cyclopentylsulfanyl-2-cyclopentylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 4.31-4.25 (q,        J=7.13 Hz, 2H), 3.36-3.16 (dd, J=14.7 Hz, 2H), 2.11-1.98 (m,        2H), 1.75-1.55 (m, 16H), 1.32 (t, J=7.15 Hz). MS (ESI) m/z: 387        (M+H⁺, 35), 409 (M+Na⁺, 100).    -   3-Butylsulfanyl-2-butylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 4.28-4.26 (q,        J=7.16 Hz, 2H), 3.33-3.14 (dd, J=14.8 Hz, 2H), 3.18-3.29 (m,        2H), 2.61 (t, J=7.27 Hz, 2H), 1.64-1.29 (m, 11H), 0.95-0.88 (tt,        6H). MS (ESI) m/z: 363 (M+H⁺, 75), 385 (M+Na⁺, 100).    -   4-Hydroxy-3-isobutylsulfanyl-2-isobutylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 4.33-4.14 (m,        2H), 3.33-3.14 (dd, J=14.8 Hz, 2H), 3.10 (m, 2H), 2.51 (d, J=6.9        Hz, 2H), 1.89-1.75 (m, 2H), 1.31 (t, J=7.1 Hz, 3H), 1.03-0.95        (m, 12H). MS (ESI) m/z: 385 (M+Na⁺, 100).    -   4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.91-7.54 (m,        8H), 7.55-7.48 (m, 6H), 4.00 (m, 2H), 1.15 (t, 3H). MS (ESI)        m/z: 503 (M+H⁺, 100) and 525 (M+Na⁺, 70).    -   4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (DMSO-d₆, 300.16 MHz) δ (ppm):        7.71-7.59 (m, 10H), 4.25 (d, J=13.8 Hz, 1H), 4.07-3.84 (m, 3H)        and 1.01 (t, J=7.1 Hz, 3H). ¹³C NMR (DMSO-d₆, 75.48 MHz) δ        (ppm): 170.11, 168.99, 160.98, 155.44, 154.61, 133.84, 133.46,        131.27, 130.97, 130.60, 130.57, 125.28, 124.85, 83.94, 62.16 and        14.17. MS (ESI) m/z: 539 (M+H⁺, 100).    -   4-Hydroxy-5-oxo-3-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm) 7.94-7.87        (m, 4H), 7.47-7.40 (m, 6H), 4.20-4.08 (m, 2H), 4.03-3.90 (m, 2H)        and 1.05 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm)        168.26, 158.45, 129.99, 129.71, 128.67, 128.56, 126.16, 126.06,        86.84, 62.19, 37.53 and 12.75. MS (ESI) m/z: 537 (M+H⁺, 100).    -   4-Hydroxy-3-(4-methoxy-benzylsulfanyl)-2-(4-methoxy-benzylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.29-7.22 (m,        4H), 6.88-6.83 (m, 4H), 4.38 (q, J=12.7 Hz, 2H), 4.23-4.12 (m,        2H), 3.81 (s, 3H), 3.79 (s, 3H), 3.76-3.71 (m, 2H), 3.17 (d,        J=15.0 Hz, 1H), 2.79 (d, J=15.0 Hz, 1H), 1.26 (t, J=6.5 Hz, 3H).        ¹³C-NMR δ (ppm): 168.1, 167.1, 159.5, 159.1, 140.9, 130.74,        130.69, 129.8, 129.0, 128.5, 114.5, 114.4, 88.4, 63.4, 55.7,        37.4, 36.3, 35.3, 14.6, 14.4.    -   4-Hydroxy-5-oxo-3-(thiazol-2-ylsulfanyl)-2-(thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (D₂O, 300 MHz) δ (ppm): 7.65-7.59 (m,        2H), 7.20-7.11 (m, 2H), 4.07 (d, J=14.3 Hz, 1H), 3.92-3.78 (m,        4H), 3.07 (q, J=7.2 Hz, 1.0H), 1.21 (t, J=7.2 Hz, 1.7H), 1.03        (t, J=7.1 Hz, 3H). MS (ESI) m/z: 417 (M+H⁺).    -   3-Benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.38-7.23 (m,        10H), 4.48-4.37 (m, 2H), 4.26-4.12 (m, 2H), 3.81 (d, J=3.4 Hz,        2H), 3.17 (d, J=15.0 Hz, 1H), 2.72 (d, J=15.0 Hz, 1H), 1.27 (t,        J=6.5 Hz, 3H). ¹³C-NMR δ (ppm): 167.9, 166.7, 140.8, 137.6,        136.9, 129.3, 129.1, 128.8, 128.7, 127.9, 127.8, 127.3, 88.1,        63.1, 37.6, 35.9, 35.3, 14.1.    -   4-Hydroxy-3-(4-methoxy-phenylsulfanyl)-2-(4-methoxy-phenylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.36 (d,        J=8.8 Hz, 2H), 7.32 (d, J=8.9 Hz, 2H), 6.87 (d, J=8.8 Hz, 4H),        3.93-3.81 (m, 2H), 3.79 (s, 3H), 3.78 (s, 3H), 3.55 (s, 2H) and        1.12 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm):        167.32, 167.04, 160.22, 159.76, 146.03, 134.30, 133.88, 125.88,        122.44, 121.01, 114.41, 114.39, 86.29, 62.46, 54.60, 54.52,        41.09 and 12.81. MS (ESI) m/z: 463 (M+H⁺, 100) and 485 (M+Na⁺,        85).    -   3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.54-7.51        (m, 1H), 7.45-7.39 (m, 2H), 7.32-7.18 (m, 5H), 3.96-3.75 (m, 4H)        and 1.07 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm):        167.00, 166.22, 149.19, 135.37, 133.96, 133.16, 132.37, 130.96,        130.02, 129.72, 129.65, 128.30, 128.11, 127.28, 127.16, 86.22,        62.71, 37.82 and 12.68. MS (ESI) m/z: 471 (M+H⁺ 100) and 493        (M+Na, 98).    -   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.90-7.78        (m, 4H), 7.49-7.42 (m, 2H), 7.37-7.31 (m, 2H), 4.50 (d, J=14.7        Hz, 1H), 4.29 (d, J=14.7 Hz, 1H), 4.12-3.97 (m, 2H) and 1.09 (t,        J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm): 167.07,        165.68, 153.35, 152.80, 135.86, 135.71, 126.63, 126.48, 125.21,        124.98, 121.65, 121.56, 121.39, 85.78, 63.28, 37.27 and 13.07.        MS (ESI) m/z: 517 (M+H⁺).    -   3-(Benzooxazol-2-ylsulfanyl)-2-(benzooxazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 7.60-7.48        (m, 4H), 7.36-7.25 (m, 4H), 4.38 (d, J=14.8 Hz, 1H), 4.33 (d,        J=14.8 Hz, 1H), 4.14-4.05 (m, 2H) and 1.16 (t, J=7.1 Hz, 3H).        ¹³C NMR (CD₃OD, 75.48 MHz) δ (ppm): 166.73, 165.39, 163.72,        160.13, 152.29, 152.20, 151.52, 141.54, 141.43, 125.19, 125.08,        124.83, 124.72, 118.76, 118.40, 110.71, 110.27, 110.12, 85.46,        63.42, 36.28 and 13.09. MS (ESI) m/z: 485 (M+H⁺).    -   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 8.90 (d,        J=4.9 Hz, 1H), 8.82 (d, J=4.9 Hz, 1H), 7.54 (d, J=4.9 Hz, 1H,        7.52 (d, J=4.9 Hz, 1H), 4.26 (d, J=14.4 Hz, 1H), 4.15 (d, J=14.4        Hz, 1H), 4.15-3.95 (m, 2H) and 1.11 (t, J=7.1 Hz, 3H). ¹³C NMR        (CD₃OD, 75.48 MHz) δ (ppm): 173.33, 172.43, 171.76, 168.57,        160.22, 160.10, 158.01, 155.51 (q, J=36.2 Hz), 155.20 (q, J=36.5        Hz), 120.35, (q, J=274.8 Hz), 120.30, (q, J=274.7 Hz), 112.58,        112.37, 101.11, 85.90, 61.86, 35.32 and 12.75. MS (ESI) m/z: 543        (M+H⁺, 26) and 565 (M+Na⁺, 100).    -   4-Hydroxy-3-(4-methyl-pyrimidin-2-ylsulfanyl)-2-(4-methyl-pyrimidin-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 8.62 (d,        J=4.9 Hz, 2H), 8.55 (d, J=4.9 Hz, 2H), 7.23 (t, J=4.9 Hz, 1H),        7.22 (t, J=4.9 Hz, 1H), 4.24 (d, 1H), 4.09 (d, 1H), 4.10-3.85        (m, 2H) and 1.07 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ        (ppm): 171.87, 171.56, 170.56, 168.78, 157.63, 157.38, 117.50,        117.22, 103.01, 85.95, 61.95, 35.05 and 12.81. MS (ESI) m/z: 407        (M+H⁺).    -   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;        ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm) 7.45-7.40 (m, 2H), 7.38-7.33        (m, 2H), 7.20-7.10 (m, 4H), 3.90 (s, 2H), 3.88 (d, J=14.3 Hz,        1H) and 3.79 (d, J=14.3 Hz, 1H). ^(13C) NMR (CD3OD, 75.48 MHz) δ        (ppm) 172.73, 158.01, 151.24, 149.97, 139.29, 138.92, 122.38,        122.29, 113.96, 113.76, 89.04, 63.38 and 36.58. MS (ESI) m/z:        441 (M+H⁺).    -   4-Hydroxy-3-(4-methyl-pyrimidin-2-ylsulfanyl)-2-(4-methyl-pyrimidin-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm): 8.42 (d,        J=5.1 Hz, 1H), 8.37 (d, J=5.1 Hz, 1H), 7.08 (d, J=5.1 Hz, 1H),        7.06 (d, J=5.1 Hz, 1H), 4.20 (s, 2H), 4.12-4.02 (m, 1H),        4.00-3.90 (m, 1H), 2.49 (s, 3H), 2.46 (s, 3H) and 1.09 (t, J=7.1        Hz, 3H). ¹³C NMR (CD3OD, 75.48 MHz) δ (ppm): 170.05, 169.73,        168.60, 168.15, 168.04, 167.24, 157.04, 156.72, 117.10, 116.67,        86.03, 61.98, 34.71, 22.50, 22.49 and 12.74. MS (ESI, negative)        m/z: 435 (M+H⁺).    -   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm) 8.62 (d,        J=4.9 Hz, 2H), 8.55 (d, J=4.9 Hz, 2H), 7.23 (t, J=4.9 Hz, 1H),        7.22 (t, J=4.9 Hz, 1H), 4.24 (d, 1H), 4.09 (d, 1H), 4.10-3.85        (m, 2H) and 1.07 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD, 75.48 MHz) δ        (ppm) 171.87, 171.56, 170.56, 168.78, 157.63, 157.38, 117.50,        117.22, 103.01, 85.95, 61.95, 35.05 and 12.81. MS (ESI) m/z: 407        (M+H⁺, 12) and 429 (M+Na⁺, 100).    -   4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (D₂O, 300.16 MHz) δ (ppm) 4.22 (q,        J=7.1 Hz, 2H), 3.43 (d, J=14.9 Hz, 1H), 3.22-2.86 (m, 9H) and        1.22 (t, J=7.1 Hz, 3H). ¹³C NMR (D₂O, 75.48 MHz) δ (ppm) 170.38,        169.75, 149.25, 117.51, 87.18, 64.13, 51.37, 51.12, 36.82,        27.57, 26.58 and 13.16. MS (ESI, negative) m/z: 232 (M²−/2,        100), 465 (M²−+H, 22) and 487 (M²−+Na+, 39).    -   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (DMSO-d6, 300.16 MHz) δ (ppm) 8.90 (d,        2H), 8.36-8.29 (m, 4H), 7.96-7.88 (m, 3H), 7.38 (s, 1H), 4.23        (d, 1H), 4.12 (d, 1H), 3.89 (broad m, 2H) and 0.93 (t, J=6.3 Hz,        3H). ¹³C NMR (DMSO-d6, 75.48 MHz) δ (ppm) 167.44, 166.76,        151.63, 146.89, 146.49, 145.90, 130.47 (q, J=32.1 Hz), 130.42        (q, J=32.2 Hz), 128.23, 127.88, 127.69, 126.09, 126.01, 122.58,        120.88, 85.74, 63.07, 35.91 and 13.92. MS (ESI) m/z: 641 (M+H⁺).    -   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.81-7.58 (m,        4H), 7.44-7.08 (m, 4H), 4.23 (d, J=13.3 Hz, 2H), 3.91 (m, 2H),        0.94 (m, 3H). MS (ESI) m/z: 613 (M+H⁺).    -   4-Hydroxy-5-oxo-3-(5-sulfonic        acid-1H-benzoimidazol-2-ylsulfanyl)-2-(5-sulfonic        acid-1H-benzoimidazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (D₂O, 300.16 MHz) δ (ppm) 7.86 (s, 1H),        7.81 (s, 1H), 7.56 (td, J=8.5 & 1.3 Hz, 2H), 7.41 (d, J=8.5 Hz,        2H), 4.11 (d, J=14.9 Hz, 1H), 4.01 (d, J=14.9 Hz, 1H), 3.70-3.60        (m, 1H), 3.48-3.38 (m, 1H) and 0.69 (t, J=7.1 Hz, 3H). ¹³C NMR        (D2O, 75.48 MHz) δ (ppm) 171.44, 169.44, 159.68, 153.04, 151.84,        136.72, 120.04, 119.93, 113.81, 112.11, 111.55, 99.01, 85.50,        63.67, 37.48 and 12.38. MS (ESI, negative) m/z: 641 (M−H⁺)    -   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H NMR (CD₃OD, 300.16 MHz) δ (ppm) 4.34 (d,        J=14.1 Hz, 1H), 4.11 (d, J=14.1 Hz, 1H), 4.10 (q, J=7.1 Hz, 2H),        3.87-3.79 (m, 6H), 3.66 (t, J=6.4 Hz, 2H), 2.16-2.05 (m, 4H),        2.03-1.92 (m, 4H) and 1.24 (t, J=7.1 Hz, 3H). ¹³C NMR (CD₃OD,        75.48 MHz) δ (ppm) 192.30, 191.83, 173.94, 169.76, 159.38,        106.58, 87.36, 63.77, 56.76, 52.41, 51.95, 43.44, 27.60, 27.14,        25.48, 25.30 and 14.39. MS (ESI) m/z: 477 (M+H⁺).    -   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 4.28 (q,        J=7.14 Hz, 2H), 3.90 (m, 1H), 3.19 (dd, J=14.7 Hz, 2H), 2.81 (m,        1H), 2.09-1.31 (m, 23H). MS (ESI) m/z: 415 (M+H⁺, 60).    -   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester, hydrochloride salt; ¹H NMR (D₂O, 300.16 MHz):        4.20 (qd, J=7.1 & 1.3 Hz, 2H), 3.50-3.21 (m, 8H), 2.90 (q, J=7.6        Hz, 2H), 2.81-2.79 (m, 12H) and 1.17 (t, J=7.1 Hz, 3H) ppm.        ^(13C) NMR (CDCl₃, 75.48 MHz): 168.13, 167.17, 142.16, 124.07,        87.07, 64.53, 56.97, 56.33, 43.07, 42.79, 42.72, 36.59, 27.25,        24.78 and 13.24 ppm. MS: m/z=393 (M+H⁺).

Example 34

By following the procedures in the preceding examples and as describedin reaction Scheme 3, the following compounds of Formula III wereprepared:

-   -   4-(Benzothiazol-2-ylsulfanyl)-5-benzoyl-3-hydroxy-5H-furan-2-one;        ¹H NMR ((CD₃)₂SO, 300.16 MHz) δ (ppm): 7.95-8.04 (m, 3H),        7.35-7.70 (m, 6H), 7.12 (s, 1H). ¹³C NMR ((CD₃)₂SO, 75.04 MHz)        δ: 192.6, 167.0, 163.0, 153.2, 147.1, 135.7, 135.1, 129.6,        129.4, 127.0, 125.5, 122.5, 122.0, 115.0, 78.7. MS (ESI-Pos)        m/z: 370.0 (M+H⁺).    -   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic        acid diethyl ester; ¹HNMR (300 MHz, d₆-DMSO-D₂O) δ=7.50-7.45 (m,        2H), 7.20-7.15 (m, 2H), 4.20-4.05 (m, 4H), 1.15-1.00 (m, 6H)        ppm. ¹³C NMR (75 MHz, d₆-DMSO-D₂O) δ=165.9, 164.5, 150.3, 147.2,        138.7, 123.0, 114.6, 109.1, 84.4, 63.8, 14.0 ppm. MS (API-ES)        m/z 393 (M+H⁺).    -   5-Acetyl-4-(benzothiazol-2-ylsulfanyl)-3-hydroxy-5H-furan-2-one        ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.88-7.78 (m, 2H), 7.53-7.34        (m, 2.3H), 5.10 (s, 1H), 2.25 (s, 3H). ¹C-NMR δ (ppm): 202.8,        134.6, 127.5, 126.2, 121.6, 121.4, 81.5, and 25.2. MS (ESI): 308        (M+H⁺), 525.    -   3-Benzylsulfanyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic        acid ethyl ester; ¹H-NMR (CDCl₃, 300 MHz) δ (ppm): 7.32 (m, 5H),        5.13 (s, 1H), 4.41-4.24 (q, 2H), 4.24-4.16 (m, 2H), 1.28 (t,        3H). MS (ESI) m/z: 295 (M+H⁺, 100), 317, (M+Na⁺, 70).    -   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic        acid 2-isopropyl-5-methyl-cyclohexyl ester; ¹H-NMR (CDCl3, 300        MHz) δ (ppm): 7.42 (d, J=8.2 Hz, 1H), 7.33 (s, 1H), 6.94 (d,        J=8.2 Hz, 1H), 5.31 (m, 1H), 4.75 (m, 1H), 2.40 (s, 3H),        1.86-1.60 (m, 4H), 1.40-1.36 (m, 2H), 0.96-0.63 (m, 12H). MS        (ESI) m/z: 445 (M+H⁺).

Example 35 Neuronal Ischemia Stress Assay

A. Isolation and Culture of Primary Hippocampal Neuronal Cells.

Materials

-   -   Neurobasal/B27: Neurobasal medium (Invitrogen, Carlsbad, Calif.)        with 1× B27 supplement (Invitrogen Life Technologies), 0.5 μM        L-glutamine, 25 μM L-glutamic acid, and 1×        Penicillin/Streptomycin.    -   Hank's Basic Salt Solution (HBSS, Ca/Mg-free) was prepared by        preparing 1× Hanks CMF (Gibco) supplemented with HEPES (10 mM,        pH 7.3), sodium bicarbonate (0.35%), 1× Penicillin/Streptomycin,        and 1 mM pyruvate.    -   Poly-D-lysine (Sigma, St. Louis, Mo.), 50 μg/ml solution        filtered through 0.2 μm filter tubes.    -   Sigmacote (Sigma, St. Louis, Mo.).    -   Plastic Culture Flasks (T75 cm²) or 12-well cell culture plates        treated with Poly-D-Lysine (Sigma, St. Louis, Mo.).        Preparation of Primary Hippocampal Neuronal Cells

A pregnant female mouse (E18-E19) was euthanized with CO₂ prior toremoval of the uterus, which was then placed in a sterile plastic petridish. The embryos were removed from the sac, and the embryonic brainswere removed and immersed in cold (4° C.) Buffered Salt Solution (HBSS;Ca/Mg free; Invitrogen Life Technologies) in a small petri dish.Hippocampi were then removed from the brains under a dissectingmicroscope and were placed on a paraffin-covered dish. The meninges werestripped away and the dissected hippocampi were collected in a smallpetri dish in HBSS. The hippocampi were transferred to a 15-mlcentrifuge tube (normally 10-12 brains)filled with HBSS. The tubecontaining the brains was centrifuged at 1000 rpm for 2 min in atabletop centrifuge. The supernatant was removed, 2 ml of HBSS was addedto the hippocampi in the tube, and the resulting suspension wastriturated 2 times each with long-tipped siliconized glass pipetteshaving progressively smaller apertures, starting with a pipette with astandard size opening (approximately 1.0 mm diameter), following withone having an aperture of half standard size (approximately 0.5 mmdiameter), then with one having an aperture about one-half that size(0.25 mm diameter). The suspension was then centrifuged again at 1000rpm for 2 min in a tabletop centrifuge, the supernatant was discarded,and 2 ml of Neurobasal/B27i (with antibiotics) was added to the tube.The trituration procedure described above was then repeated on thissuspension.

The density of cells was determined on a small aliquot of cells usingstandard counting procedures and correcting for cell viability by trypanblue stain exclusion. Using this procedure, the expected yield is3×10⁵-6×10⁵ cells/brain. Cells were then added to PDL-coated 24-wellplates, flasks or MetTek dishes in Neurobasal/B27I at a density of about1.5×10⁶ cells (T75 flask) or about 70,000 cells/well of a 24-well plate.Plated cells were incubated at 37 degrees in an atmosphere of 5% CO₂/95%O₂. Media was renewed after 3-4 days by replacing half of it with freshNeurobasal/B27m medium, containing 5 μM cytosine arabinoside (AraC).Seven to eight days from the initial culture, the media was renewedagain, by removing one-half or it and replacing with an equal amount offresh Neurobasal/B27m medium (without Ara-C).

B. Hippocampal Anoxia-Reoxygenation Cell Death Assay.

This assay was used to induce ischemia by anoxia-reoxygenation incultured hippocampal neuronal cells. Test compounds were added to assesspotency and efficacy against ischemia-induced neuronal cell injury andcell death.

Materials.

-   -   Neurobasal media, NoG neurobasal media, B27 supplement and B27        Supplement minus AO were obtained from Invitrogen Life        Technologies.    -   Neurobasal/B27 medium was prepared with 2× B27 minus AO        supplement, 0.5 mM L-glutamine and 0.25×        penicillin/streptomycin.    -   Cell Tracker Green was obtained from Molecular Probes and a        fresh 5 μM solution was prepared from 10 mM stock just before        use.    -   LoG-Neurobasal contains NoG neurobasal medium plus 1 mM glucose,        0.5 mM L-glutamine, 0.25× Penicillin/Streptomycin, and 10 mM        Hepes (pH 7.4).    -   Primary hippocampal neuronal cells were prepared according to        the methods described above and were cultured in poly-D-lysine        coated 24-well plates for 10-11 days prior to use.

Deoxygenated LoG-Neurobasal medium (100 ml) was prepared bypre-equilibrating the medium in a T150 cm² flask in a hypoxic chamberovernight. Following pre-incubation under hypoxic conditions, theLoG-Neurobasal media was lightly bubbled with 100% N₂ for 30 min tocompletely deoxygenate the media. An additional 20 ml LoG-Neurobasal waspre-equilibrated in a T75 cm² flask and was incubated in a normalincubator (5% CO₂) overnight. Reoxygenated medium was prepared byplacing Neurobasa/B27 media overnight in the culture incubator (5%CO₂/95% O₂).

10-11 Days after plating the hippocampal neurons, existing culturemedium (Neurobasal/B27m) was removed from the cells by aspiration. Cellswere washed once with 600 μl/well (24-well culture plates) of glucosefree-BSS. Neurons were replenished with deoxygenated LoG-Neurobasal (400μl per well for each well of a 24-well plate). Test compounds were addeddirectly to each well (usually 3 concentrations of the compound pluspositive control, each in triplicate). Most test compounds weredissolved in 100% DMSO; however, concentrations were adjusted such thatthe final concentration of DMSO in the cell media never exceeded 0.5%.Plates containing cells with test compounds were placed in a hypoxicchamber for 4-5 hr with plate lids ajar. For normoxia controls,pre-equilibrated normoxic LoG-Neurobasal medium was added to each wellof cells, and the plate was replaced in the normal culture incubator for4-5 hr. After 4-5 hr of hypoxia, the existing media was carefullyaspirated off, and 400 μL of new, reoxygenated (pre-equilibrated)Neurobasal/B27 was added to each well. The same test compounds (in thesame the concentrations) were added back into the corresponding wells.Plates were placed in the cell culture incubator (5% CO₂/95% O₂) andreoxygenated for 20-24 hr. After reoxygenation for 20-24 hr, liveneurons were quantitated using the cell tracker green fluorescencemethod, described below.

To test for cell viability, existing culture medium was aspirated fromeach well of the 24 well plates, and neurons were washed once with 1 mLof HBSS (pH 7.4, pre-warmed to 30-37° C.). To each well was added 500 μLof 5 μM Cell Tracker Green fluorescent dye dissolved in HBSS. Plateswere placed in the dark at room temperature for 15 minutes, then werewashed with 1 mL of HBSS. 500 μL of HBSS was then added to each well,and fluorescent cells were counted using a fluorescent microscope.Significantly increased cell viability compared to control cells isindicative of a protective compound.

Compounds of the present invention when tested as described aboveprovide protection against stressor-induced cell death in at least about20% of the cells tested, at concentrations ranging from about 1 to 1000μM.

When tested as described above, compounds of the present invention, suchas

-   4-(Benzothiazol-2-ylsulfanyl)-5-(benzothiazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   1,4-Dihydroxy-4-methyl-3a,4-dihydro-3-oxa-10-thia-4a,9-diaza-cyclopenta[b]fluoren-2-one;-   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   4-(Furan-2-ylmethylsulfanyl)-5-(furan-2-ylmethylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   5-Acetyl-4-(benzothiazol-2-ylsulfanyl)-3-hydroxy-5H-furan-2-one;-   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2,5-dihydro-furan-2-carboxylic    acid;-   3-(Furan-2-ylmethanesulfonyl)-2-(furan-2-ylmethanesulfonylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Benzylsulfanyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic acid    ethyl ester;-   3-[4-(2-Carboxy-vinyl)-phenylsulfanyl]-2-[4-(2-carboxy-vinyl)-phenylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(Benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-Hydroxy-5,6-dimethyl-2-oxo-5,6-dihydro-2H-1-oxa-4,7-dithia-azulene-8a-carboxylic    acid ethyl ester;-   4-(2,2-Dimethyl-propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-Dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanyl-methyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid 2-isopropyl-5-methyl-cyclohexyl ester;-   3-Cyclopentylsulfanyl-2-cyclopentylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Butylsulfanyl-2-butylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(9H-purin-6-ylsulfanyl)-2-(9H-purin-6-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methoxy-benzylsulfanyl)-2-(4-methoxy-benzylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(4-methoxy-phenylsulfanyl)-2-(4-methoxy-phenylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-(4-methyl-piperazine-1-carbonyl)-5H-furan-2-one;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzooxazol-2-ylsulfanyl)-2-(benzooxazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   4-Hydroxy-3-(1H-imidazol-2-ylsulfanyl)-2-(1H-imidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Diethylamino-ethylsulfanyl)-2-(2-diethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-2-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid methyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester, hydrochloride salt;-   4-Hydroxy-3-(2-methoxycarbonyl-ethylsulfanyl)-2-(2-methoxycarbonyl-ethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-methoxycarbonylmethylsulfanyl-2-methoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyridin-4-ylsulfanyl)-2-(pyridin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   5,8-Dichloro-3-hydroxy-2-oxo-2H-1-oxa-4,9-dithia-benzo[f]azulene-10a-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-amino-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-p-tolylsulfanyl-2-p-tolylsulfanylmethyl-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;    provided protection against stressor-induced cell death in at least    about 40% of the cells tested, at concentrations ranging from 1 to    100 micromolar.

Example 36 Myocyte Calcium-Contractility Assay

A. Isolation and Culture of Primary Neonate Myocytes.

Materials

-   -   10× Heart Dissection Solution (HDS) contains the following        components (g/l) in cell culture grade water:_NaCl, 68; HEPES,        47.6; NaH₂PO₄, 2; Glucose, 10; KCl, 4; MgSO₄, 1, pH adjusted to        7.4. Prior to filter sterilization of diluted (1× HDS) solution,        10 mg phenol red was added to each 500 milliliters of medium.    -   Transferrin and Bovine Insulin were obtained from Invitrogen        Life Technologies (Carlsbad, Calif.), and resuspended at a        concentration of 4 mg/ml in tissue culture grade water.    -   DMEM-F12-DMEM-F12, powder, 1:1 containing glutamine and        pyridoxine hydrochloride was purchased from Invitrogen Life        Technologies. To one liter equivalent of the powder was added        2.43 g of sodium bicarbonate and 10 ml of 100×        Penicillin/Streptomycin in 950 ml of tissue culture grade water        with stirring. The pH was adjusted to 7.2 with 1 M HCl and        volume was adjusted to 1 liter. The solution was filter        sterilized then 2.5 ml of 4 mg/ml Transferrin, 250 μl 4 mg/ml        Insulin and 30.7 mg of bromodeoxyuridine were added.    -   DMEM-F12 was also prepared with 4% fetal bovine serum (FBS) for        pre-coating the tissue culture plates and initial suspension of        the cardiomyocyte pellet.    -   Collagenase solution-49 mg of collagenase was resuspended in 120        ml 1× HDS.    -   Fetal Bovine Serum (FBS) from Invitrogen Life Technologies        Preparation of Primary Neonatal Myocyte Cultures

Tissue culture ware was pre-coated with DMEM-F12-4% FBS by incubating 50μl per well of a 96-well plate and 0.25 ml per 12-well plate at 37° C.

Two-day old rat pups were removed from their mothers and placed in asterile container. Pups were dipped quickly into 70% alcohol, thendecapitated and the body was placed in an empty sterile tissue culturedish. An incision was made starting at the neck and progressing towardsthe belly, cutting through the sternum. The heart was removed and placedin a tissue culture dishes containing 1× HDS. The atria were trimmed,and the remaining ventricles were placed into a separate tissue culturedish containing 1× HDS, where they were sectioned into 3-4 pieces each.Ventricles were then transferred to a sterile 250 ml glass flask and the1× HDS was removed. Twenty milliliters of pre-warmed collagenasesolution were added to the ventricles, followed by incubation at 37° C.with shaking. After 30 minutes, the collagenase solution was removed andreplaced with 20 ml fresh pre-warmed collagenase. Incubation wascontinued for an additional 30 minutes. At the end of the incubation,any tissue chunks were allowed to settle prior to removing thecollagenase (containing the isolated cardiomyocytes) from the disruptedtissue pieces. The isolated myocytes were added to a 50 ml Falcon tubecontaining 2 ml Fetal Bovine Serum (FBS). The remaining tissue pieceswere subjected to a second digestion by adding 20 ml fresh pre-warmedcollagenase and incubating as above for 30 minutes. This second digestwas then centrifuged at 1000 rpm for 10 minutes (tabletop centrifuge).The resulting supernatant was discarded, and the cell pellet wassuspended with 4 ml FBS. The resulting cell suspension was placed in theincubator at 37° C. This step was repeated several additional times toharvest additional material.

Percoll gradients were prepared by adding 2.5 ml of 10× HDS to 22.5 mlof Percoll (Invitrogen Life Technologies) with mixing (Percoll Stock).Top Gradient solution (11 ml Percoll Stock and 14 ml 1× HDS) and BottomGradient solution (13 ml Percoll Stock and 7 ml 1× HDS) were prepared.Four milliliters of the Top Gradient solution were transferred into 6×15ml sterile Falcon tubes. Three milliliters of the Bottom Gradientsolution were placed in each tube by inserting a serological pipette tothe bottom of the tube and slowly adding the liquid.

All the digests (5) were pooled in one 50 ml Falcon tube and centrifugedon a tabletop centrifuge at 1000 rpm for 10 minutes. The supernatant wasdiscarded, and the cell pellet was resuspended in 12 ml of 1× HDS. Twomilliliters of the cell suspension was added to the top of eachgradient. The gradient tubes were then centrifuged at 3000 rpm for 30minutes without braking in a Beckman Allegra 6 centrifuge (GH 3.8Arotor). Following centrifugation, the cells segregated into two sharpbands at the two interfaces. The lower band of the two bands wasenriched for cardiomyocytes; there was also a cardiomyocyte pellet atthe bottom of the tube. The upper band was enriched for fibroblasts andother non-cardiomyocytes. The upper portion of the gradient wasaspirated down to just above the cardiomyocyte layer. The cardiomyocytelayer was then carefully removed along with the pellet, and the twofractions were pooled in a sterile 50 ml Falcon tube, along withcorresponding fractions from additional gradient tube; then 1× HDS wasadded to a total volume of about 50 ml. The tube was centrifuged at 1000rpm for 7 minutes. The supernatant was discarded and resuspended in 25ml 1× HDS. A further 25 ml of 1× HDS was added and the centrifugationstep was repeated. The cell pellet was resuspended carefully butthoroughly in 40-50 of DMEM/F12-4% FBS.

A small aliquot of the cell suspension was counted in a hemocytometer.The DMEM/F12-FBS coating medium was aspirated from the tissue culturedishes. The cardiomyocytes were added to the dishes at a plating densityof 7.5×10⁴/well per 96-well in 200 μL and 1.5×10⁵/well per 12-well in 3ml. The cultures were incubated at 37° C. with 5% CO₂ overnight. Theoriginal medium was removed, and add fresh DMEM/F12-5% FBS was added toeach culture, prior to incubation at 37° C. with 5% CO₂ for a further 48hours, before use.

B. Contractility Assay

Materials

-   -   Complete DMEM-F12: DMEM/F12, powder, 1:1 containing glutamine        and pyridoxine hydrochloride was purchased from Invitrogen Life        Technologies (Carlsbad, Calif.). Powder sufficient to prepare        one liter of buffer and 2.43 g of sodium bicarbonate was mixed        into 950 ml of tissue culture grade water. The pH was adjusted        to 7.2 with 1 M HCl and the remaining water was added to make 1        liter. Following filter sterilization, 10 ml of 100×        Penicillin/Streptomycin, 2.5 ml of 4 mg/ml Transferrin, 250 μl 4        mg/ml Insulin and 30.7 mg of bromodeoxyuridine were added, and        the mixture was incubated at 37° C. prior to use.    -   1 mM glucose in DMEM was made from DMEM without L-glutamine,        without glucose, without sodium pyruvate, purchased from        Invitrogen Life Technologies.    -   20 μM Fluo-4: Cell permanent AM ester of Fluo-4 was obtained        from Molecular Probes (Eugene, Oreg.) as a dry powder to be        stored at −20° C. This fluorescent dye is light sensitive and        should be made up fresh at 1 mM in DMSO prior to use to prevent        light degradation.    -   10 mM CaCl₂ solution was made fresh each day in 1× HBSS and        incubated at 37° C. prior to use.

Neonatal cardiomyocytes were isolated as described above. Thecardiomyocytes were plated in 96-well format (black clear-bottomedplates) at a density of 7.5×10⁴ per well and grown for 2 days in thepresence of 5% FBS prior to use in the assay.

Physiological ischemia was simulated by placing the cardiomyocytes in ananaerobic chamber (0% O₂, 85% N₂, 5% CO₂ & 10% H₂) in DMEM containing 1mM glucose. Positive control cells are treated with DMEM-F12 containing25 mM Glucose, which protects against the anoxia.

The test compounds were made up in DMEM-1 mM glucose in 96 deep-wellmother plates and appropriately diluted for use in the assay. The mediawas removed from the cells and replaced with 200 μl of either DMEM-F12or 1 mM DMEM with or without test compounds. The plates were then placedinside the 37° C. incubator in the anaerobic chamber and incubated for16 hours. The plates were then removed and reoxygenated by the additionof DMEM-F12. The DMEM with or without test compounds is carefullyremoved from the cells and replaced with pre-warmed DMEM-F12 containing5% FBS. Since the anoxic treatment may damage and/or kill the cells,causing them to dislodge from the bottom of the wells gentle aspirationof media is required at this step. The cells were then placed in anormal incubator at 37° C. and incubated for two hours to allow thecells to reoxygenate.

A working solution of 20 μM Fluo-4 was added to pre-warmed 1× HBSS. Thecells were loaded with Fluo-4 by first removing media from the cells andreplacing with 100 μl of 20 μM Fluo-4. Unloaded control cells weretreated in parallel with 1× HBSS alone. All cells were then incubated at37° C. for 30 minutes. Before fluorescence measurements were made, thecells were washed in indicator-free medium (HBSS) to remove any dye thatis non-specifically associated with the cell surface. Cells were thenincubated for an additional 20 minutes at room temperature. Basal Fluo-4fluorescence was measured using the 485 nm excitation and 538 nmemission filter pair on a microplate flourometer (Fluorskan™, ThermoLabsystems Oy, Helsinki, Finland). Each well was read for 160 ms toobtain a baseline reading, then stimulated to contract by addition of 10mM CaCl₂. Following incubation at 37° C. for 30 minutes, a stimulatedfluorescence reading was taken after 90 minutes.

Compounds of the present invention when tested as described above suchas:

-   3-[3-(2-carboxy-pyrrolidin-1-yl)-2-methyl-3-oxo-propyldulfanyl]-2-[3-(2-carboxy-pyrrolidin-1-yl)2-methyl-3-oxo-propylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methoxy-1H-benzoimidazol-2-ylsulfanyl)-2-(5-methoxy-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-nitro-1H-benzoimidazol-2-ylsulfanyl)-2-(5-nitro-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-amino-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;    showed the presence of calcium transients in amounts (about 30% or    more) indicative of ability guard against ischemic damage and allow    the cells to maintain their contractile function.

Example 37 Rat Middle Cerebral Artery Occlusion (MCAO) Model of CerebralIschemia

A. Animal Preparation

Male Wistar rats (Harlan, Ind.) weighing 300-350 g are commonly used inthese experiments. Animals are allowed free access to water andcommercial rodent diet under standard laboratory conditions. Roomtemperature is maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals are acclimatized to the laboratoryenvironment 5 to 7 days prior to the study, and fasted (with free accessto water) overnight before surgery.

B. Middle Cerebral Artery Occlusion (MCAO)

Anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane,Front Dodge, Iowa) in 0.8% oxygen. The animal's neck was shaved andsterilized before operation. Body temperatures were controlled andmaintained at 37.5° C.+/−1 degree via external heating and coolingdevices. To lower the body temperature, animals are placed in a coolingchamber, which uses ice to cool circulating air. Throughout the studythe body temperature is recorded using a temperature transponder (BMDSInc., Seaford, Del.) implanted subcutaneously at the time of MCAObetween the rat shoulder blades that allows the user to read the bodytemperature via a pocket scanner (BMDS Inc., Seaford, Del.). The bodytemperature may also be taken by inserting the temperature probe intothe animal's rectum. Body temperature is recorded every hour for 6 hourspost-occlusion; however, body temperatures were taken more frequently sothat they could be maintained at the normothermic temperature.

Animals were subjected to two hours MCAO using a modified intraluminalfilament technique, as follows: A midline incision on the ventral partof the neck is made to expose external and internal carotid arteries.The right external and common carotid arteries are ligated by a suture(silk 5/0, Carlisle Laboratories, Farmers Branch, Tex.) and the rightinternal artery is temporarily ligated using a microvascular clip (FineScience Tool Inc., Foster City, Calif.). A small incision was made inthe common carotid artery. A nylon filament, its tip rounded by heating,is prepared from a fishing line (Stren Fishing Lines, Wilmington, Del.)and is inserted from the right common carotid artery. The filament isadvanced into the internal carotid artery 18-20 mm from the point ofbifurcation of internal and external arteries and a suture is tightlyligated around the filament. Two hours post occlusion, animals arere-anesthetized to allow reperfusion for the remaining of the experimentby removal of the filament.

C. Drug Administration

Test compounds may be administered by any of a number of routes, such asthose described below. Compounds can be administered before, during orafter occlusion, as appropriate to the protocol.

a) Intracerebroventricular (ICV) Infusion

The anesthetized animal is placed on a stereotaxic apparatus (HarvardApparatus, S. Natick, Mass.). Anesthesia is maintained by inhalation of3.0% isoflurane (Aerrane, Front Dodge, Iowa) in 0.8% oxygen throughoutthe entire procedure. The scalp is shaved and sterilized prior tosurgery. A midline sagittal incision about 3 cm long is made slightlybehind the eyes to expose the skull. The skull is scraped with a roundedend spatula to remove periosteal connective tissue. A bur hole is placed1.5 mm lateral, 1 mm posterior to the left of the bregma to mark theleft lateral ventricle. A brain infusion cannula (ALZET Co., Palo Alto,Calif.) is inserted 4 mm deep into the hole. The desired depth isadjusted by attaching spacers to the cannula. The cannula attached to a4-cm silastic catheter (Helix Medical Inc., Carpinteria, Calif.) fixedin place with dental cement (Ketac-cement, Norristown, Pa.). Thecatheter is either attached to a primed osmotic pump placedsubcutaneously between the shoulder blades for permanent infusion or toa syringe for a short infusion.

b) Intravenous (IV) Osmotic Pump Implantation into the Jugular Vein

Anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane,Front Dodge, Iowa) in 0.8% oxygen throughout the entire procedure. Theanimal's neck will be shaved and sterilized before operation. A midlineincision is made on the ventral part of the neck to exposes the jugularvein. The vein is isolated and ligated with a suture (silk 5/0, CarlisleLaboratories, Farmers Branch, Tex.) rostral to the point of the incisionand a microvascular clip (Fine Science Tool Inc., Foster City, Calif.)close to the heart. A small incision is made between two ligations. A2-cm silastic catheter (Helix Medical Inc.) attached to a PE-60 tube(Becton. Dickinson and Co. Sparks, Md.) connected to an ALZET (ALZET CO.Palo Alto, Calif.) pump is introduced and advanced 2 mm into the jugularvein toward the heart. The microvascular clip is removed and thecatheter is secured in place with a suture (silk 5/0, CarlisleLaboratories, Farmers Branch, Tex.). The pump is placed into a pocketmade subcutaneously between the shoulder blades, allowing the catheterto reach over neck to the jugular vein with sufficient slack to permitfree movement of neck and head.

c) IV Infusion Via Femoral Vein

Anesthesia is maintained by inhalation of 3.0% isoflurane (Aerrane,Front Dodge, Iowa) in 0.8% oxygen throughout the entire procedure. Theexterior site of the right femoral vein is shaved and sterilized priorto surgery. A 3-cm incision is made in the right groin region and thefemoral vein is isolated. A small incision is made on the femoral veintemporarily ligated with a microvascular clip to introduce and advance apolyethylene (PE-50) catheter (Becton Dickinson and Co. Sparks, Md.).The catheter is secured in place with suture (silk 5/0, CarlisleLaboratories, Farmers Branch, Tex.). The other end of the catheter isattached to a syringe filled with the heparinized saline for the bolusinjection. Using a hemostat, a pocket is made subcutaneously on the backof the animal so the PE catheter can be brought up to theexteriorization point at the nape of the neck for either a bolusinjection or a continuous injection by an osmotic pump.

d) Intraperitoneal (IP) Injection

An awake rat is held in a standard hand hold position, a 23¾G needle isinjected into the lower right quarter of the abdomen pass theperitoneum, slightly off the midline. To avoid organ injection, theplunger of the syringe is slightly pulled back. If no fluid iswithdrawn, the content of the syringe is delivered into the abdominalcavity.

e) Gavage Feeding

A standard rat gavage tube (Popper & Sons Inc., NY) is attached to a3-cc hypodermic syringe. The animal is held by the shoulder in avertical position. The feeding tube is placed into the mouth thenadvanced until it reaches the stomach (the approximate insertion lengthof the tube was measured prior to the feeding). The content of thesyringe is slowly delivered, and then the tube is withdrawn.

D. Behavioral Assessment

One hour after MCAO, the animal was gently held by its tail and observedfor forelimb flexion. Then the animal is placed on the floor to beobserved for walking pattern; only the animals that score 3 on Bedersongrading system (Table 1) are included in the study.

TABLE 1 Bederson Grading System for Neurological Evaluation Neurologicaldeficit Grading Behavioral observation Normal grade 0: No observabledeficit Moderate grade 1: forelimb flexion Severe grade 2: forelimbflexion, decreased resistance to lateral push grade 3: forelimb flexion,decreased resistance to lateral push, circle to paretic sideE. Evaluation of Ischemic Damage

Twenty-four hours post-MCAO, or longer, in some experiments, animalswere sacrificed by CO₂ asphyxiation (dry ice). The brain was quicklyremoved from the skull, using standard procedures, rinsed in chilledsaline solution, and placed on a rat brain tissue slicer (ASIinstrument, MI). Seven 2-mm thick coronal slices are cut from each brainusing razor blades. The slices were immersed in 0.9% saline containing1.0% 2,3,5-triphenyltetrazolume chloride (TTC) (Sigma Chemical Co., St.Louis, Mo.) and incubated in a 37° C. water bath for 30 minutes.

After staining, each 2-mm slice is photographed with a TMC-7 camera (JHTechnologies, Ca) which is directly connected to a desktop PC to captureand save the image of each brain slice. This image is used for themeasurements of the regions of interest using a computer-based imageprocessing system (Metamorph).

To measure each area, the region of interest is selected using afreehand selection tool, the area is automatically computed by selectingthe measure command. The measurements for primary regions of interestare right hemisphere, left hemisphere, total infarct, subcorticalinfarct, total penumbra and subcortical penumbra. After all regions ofinterest are measured for all seven slices of the brain, they are sortedby slice number and the corresponding regions of interest using a custommade Excel™ macro. This macro calculates the cortical penumbra, corticalinfarct and total ischemic damage for each slice; the correspondingareas of each rat brain will be added together to produce a singlemeasurement for each area. Since the ipsilateral hemisphere is swollenfollowing MCAO, edema volume is calculated and reported as thevolumetric differences between the right and left hemispheres of eachbrain slice. Using the % of hemispheric swelling all the volumes will becorrected for the edema.

The volume of the damage is determined using the calculations below foreach rat's brain.

M asur m nt Equati n Correct d Value(s) C rtical Penumbra (C.P.) TotalPenumbra-Subcortical Total Penumbra (T.P._(corr)) = (T.P. × Penumbra %H.S./100) C.P._(corr.) = C.P. − (C.P. × % H.S./100) S.P._(corr) = S.P. −(S.P. × % H.S./100) Cortical Infarct Total Infarct-Subcortical InfarctT.I._(corr.) = T.I. − (T.I. × % H.S./100) S.I._(corr) = S.I. − (S.I. × %H.S./100) C.I._(corr.) = C.I. − (C.I. × % H.S./100) Total IschemicDamage (T.I.D.) Total Penumbra + Total Infarct T.I.D._(corrected) =T.I.D. − (T.I.D. × % H.S./100) Total Volume (mm³) Each value ismultiplied by 2 (the thickness of the tissue). Edema Volume Thevolumetric differences between the sum of right and left hemispheresdetermines the edema volume. % Hemispheric swelling (H.S.) Edema ×100/left hemisphereF. Statistical Analysis

Sample size is chosen to achieve a 90% probability of significantresults. The measurements, which represented the same region of interestin seven slices of each rat's brain are added together to yield a singlemeasurement for total infarct, subcortical infarct, cortical infarct,total penumbra, subcortical penumbra, cortical penumbra, total ischemicdamage and edema in each animal. Group data are presented asmeans+/−SEM. Differences at the level of p<0.05 are consideredstatistically significant. Between groups comparison of each region ofinterest are carried out by unpaired student t test (between two groups)or one way ANOVA followed by post hoc Bonferroni's multiple comparisonsor by the nonparametric Dunnett's test (between control and the drugtreated groups).

Certain compounds of the present invention when tested as describedabove provided a reduction in total infarct volume of at least about 40%at doses in the range of about 10 μg/kg to about 40 mg/kg.

Example 38 Model of Myocardial Infarction: Left Coronary Ligation (Rat)

Male Sprague-Dawley weighing 250-320 g are allowed free access to waterand commercial rodent diet under standard laboratory conditions. Roomtemperature is maintained at 20-23° C. and room illumination is on a12/12-hour light/dark cycle. Animals are acclimatized to the laboratoryenvironment 5 to 7 days prior to the study and are fasted overnightprior to surgery.

Surgical Procedure for Acute Studies:

Rats are anaesthetized with Urethane (1.2-1.5 gm/kg). Core bodytemperature is maintained at 37° C. by using a heating blanket. Thesurgical area is shaved, and a ventral midline incision is made toexpose the trachea and jugular area. A catheter (PE50) is placed in thejugular for administration of compound and maintenance anesthesia. Thetrachea is incised and a 14-16-gauge modified intravenous catheter isinserted and tied in place as an endotracheal tube. The animal is placedin right lateral recumbency and initially placed on a Harvard ventilatorwith a tidal volume of 5-10 ml/kg. 100% O₂ is delivered to the animalsby the ventilator. ECG electrodes are placed to record a standard LeadII ECG. The surgical site is cleaned with alcohol swab, and a skinincision is made over the rib cage over the 4^(th)-5^(th) intercostalspace. The underlying muscles are dissected with care to avoid thelateral thoracic vein, to expose the intercostal muscles. The chestcavity is entered through the 4^(th)-5^(th) intercostal space, and theincision expanded to allow visualization of the heart. The pericardiumis opened to expose the heart. A 6-0 silk suture with a taper needle ispassed around the left coronary artery near its origin, which lies incontact with the left margin of the pulmonary cone, at about 1 mm fromthe insertion of the left auricular appendage. A piece of tubing isplaced over the suture to form an occluder. The coronary artery isoccluded for 30 minutes by sliding the tube towards the heart untilresistance is felt and holding it in place with a vascular clamp. TheECG is monitored for S-T changes indicative of ischemia. After 30minutes, the occluder is removed, leaving the suture in place. The ECGis monitored for the first 10 minutes of reperfusion. The rat istransferred to the pressure control ventilator for the remainder of theprotocol. The rats are ventilated by a small animal ventilator with apeak inspiratory pressure of 10-15 cm H₂O and respiratory rate 60-110breaths/min. The heart is allowed to reperfuse for 90 minutes.

Surgical Procedure for 24 Hour Study:

Rats are anaesthetized with Ketamine/Xylazine IP (95 and 5 mg/kg) andintubated with a 14-16-gauge modified intravenous catheter. Anesthesialevel is checked every 15 minutes by toe pinch. Core body temperature ismaintained at 37° C. by using a heating blanket. The surgical area isshaved and scrubbed. A ventral midline incision is made to expose thejugular vein. A catheter (PE50) is placed in the jugular foradministration of compound and maintenance anesthesia. The animal isplaced in right lateral recumbency and initially placed on a ventilatorwith a tidal volume of 5-10 ml/kg H₂O or a pressure controlledventilator with a peak inspiratory pressure of 8-15 cm H₂O andrespiratory rate 60-110 breaths/min. 100% O₂ is delivered to the animalsby the ventilator. ECG electrodes are placed to record a standard LeadII ECG. The surgical site is cleaned with surgical scrub and alcohol. Askin incision is made over the rib cage over the 4^(th)-5th intercostalspace. The underlying muscles are dissected with care to avoid thelateral thoracic vein, to expose the intercostal muscles. The chestcavity is entered through 4^(th)-5th intercostal space, and the incisionexpanded to allow visualization of the heart. The pericardium is openedto expose the heart. A 6-0 silk suture with a taper needle is passedaround the left coronary artery near its origin, which lies in contactwith the left margin of the pulmonary cone, at about 1 mm from theinsertion of the left auricular appendage. A piece of tubing is placedover the suture to form an occluder. The coronary artery is occluded for30 minutes by sliding the tube towards the heart until resistance isfelt and holding it in place with a vascular clamp. The ECG is monitoredfor S-T changes indicative of ischemia. After 30 minutes, the occluderis removed, leaving the suture in place. The ECG is monitored for thefirst 10 minutes of reperfusion. The incision is closed in three layers.The IV catheter is removed or tunneled under the skin and exteriorizedbetween the shoulder blades to allow for blood withdrawal or furtherdrug therapy. The rat is ventilated until able to ventilate on its own.The rats are extubated and recovered on a heating pad. Once awake, theyare returned to their cage(s). Animals may receive Buprenorphine(0.01-0.05 mg/kg SQ) for post-operative analgesia. After the designatedreperfusion time (24 hours) the animals are anesthetized and the heartsremoved under deep anesthesia.

Treatment Protocols

Diet

-   -   Animals are fed a custom diet prior to or after coronary        ligation. The length of treatment varies with the study. Doses        are calculated based on the average consumption of 15 gms of        feed per day for a 300 gm rat. Rat weights are monitored during        the study. Feed not consumed is weighed to estimate consumption        rates.

Gavage

-   -   Animals are dosed orally by gavage. Length and frequency of        treatment vary with the study. A standard rat gavage tube        (Popper & Sons Inc, NY) is attached to a 3-cc hypodermic        syringe. The animal is held by the shoulder in a vertical        position. The feeding tube is placed into the mouth then        advanced until it reaches the stomach (the approximate insertion        length of the tube is measured prior to the feeding). The        content of the syringe is slowly delivered, and then the tube is        withdrawn.

IV Treatment

-   -   A ventral incision is made to expose the jugular area. A        catheter (PE50) is placed in the jugular vein for administration        of compound. Animals are dosed by bolus injection and/or        continuous infusion. The time and duration of treatment varies        with the protocol.        Tissue Processing

After reperfusion, each animal receives 200 units of heparin IV undergeneral anesthesia and the heart is removed and placed in cold saline.After removal the coronary artery is ligated with the suture that isalready in place. The heart is placed on a perfusion apparatus and EvansBlue dye is infused to delineate the area at risk. The heart is then cutinto five 2-mm thick transverse slices from apex to base. The slices areincubated in 1% triphenyltetrazolium chloride (TTC) (Aldrich, Milwaukee,Wis.) in 0.9% saline for 20 minutes at 37° C. Tetrazolium reacts withNADH in the presence of dehydrogenase enzymes causing viable tissue tostain a deep red color and that is easily distinguished from theinfarcted pale-unstained necrotic tissue. The slices are placed apexside down in the lid of a small petri dish for the staining procedure.The bottom of the dish is placed over the slices to keep them flat. Theslices are photographed in order from apex to base, with the base sideup. The areas of infarcted tissue, area at risk and the whole leftventricle are determined using a computerized image analysis system. Thetotal area for each region is added together to give a total for theentire heart. Infarct size is expressed both as a percentage of thetotal ventricle and the area at risk.

Statistical Analysis

Group data is represented as means+/−SEM. Comparisons between treatmentgroups are made using ANOVA with p<0.05 considered significant. Post hoccomparisons may be made using either Dunnett's test or Tukey's test.

The compounds of the present invention showed activity when tested bythis method.

Example 39 Evaluations of Sensorimotor Behavior

A. Fore and Hindlimb Grip Strength Test in Rats

Animals with cerebral infarction induced by transient or permanentunilateral occlusion of the middle cerebral artery (MCA) andsham-operated rats are tested for grip strength, a standard model ofneuromuscular function and sensorimotor integration, using aComputerized Grip Strength Meter for Rats (Dual Stand Model, ColumbusInstruments, Columbus, Ohio).

Animals are moved into the testing room for 30 minutes before testing.Prior to testing, each gauge is calibrated with a set of known weightsand the apparatus is adjusted for the size of animal, according tomanufacturer's instructions. The forelimb measurements are carried outwith the meter in the tension peak mode to freeze the reading as thesubject is pulled away from the grip bar. The hindlimb measurements arecarried out with the meter in the compression peak mode to freeze thereading as the subject's hindlimbs are pulled over the bar toward themeter. Each animal is hand-held by the investigator as pulled past thegrip bars, using a consistent technique, leaving the fore and hind limbsfree to grasp the grip bars.

Testing is carried out on postoperative day 2 and repeated, in ablind-randomized fashion, twice weekly for a defined interval.Typically, three successive readings are taken for each animal with anintertrial interval long enough to record the data and zero both metersfor the next trail.

B. Rota-Rod Test in Rats

Apparatus: Rota-Rod Treadmill for Rats (7750 Accelerating Model, fromUGO BASILE, COMERIO-ITALY).

Procedure: Animals with cerebral infarction induced by transient orpermanent unilateral occlusion of the middle cerebral artery (MCA) andsham-operated rats are tested in this study, using a Rota-Rod Treadmillfor Rats (7750 Accelerating Model, UGO Basile, Comerio, Italy). Theanimals are moved into the testing room 30 minutes before testing. Everyrat receives 2-3 training runs of 1-2 minutes at intervals of 2-3 hoursbefore testing.

The cylinder on the apparatus is set in motion before placing the ratsin position. The motor is set at a constant selected speed in 7700 onRESET mode, and the rats are placed, one by one, in their sections.

Testing is carried out on postoperative day 2 and repeated, in ablind-randomized fashion, twice weekly for a defined interval.Typically, three successive readings are taken for each animal with anintertrial interval long enough to record the data and zero both metersfor the next trail.

The compounds of the present invention can be tested by this method.

Example 40 Model of Congestive Heart Failure

Experimental Preparation

225-275 g male Sprague-Dawley rats are anaesthetized withketamine/xylazine (95 mg/kg and 5 mg/kg) and intubated with a14-16-gauge modified intravenous catheter. Core body temperature ismaintained at 37° C. by using a heating blanket. The surgical area isclipped and scrubbed, and the animal is placed in right lateralrecumbency and initially placed on a ventilator with a peak inspiratorypressure of 10-15 cm H₂O and respiratory rate 60-110 breaths/min. 100%O₂ is delivered to the animals by the ventilator. ECG electrodes arepositioned to record a standard Lead II ECG. An incision is made overrib cage over the 4th-5th intercostal space. The underlying muscles aredissected with care to avoid the lateral thoracic vein, to expose theintercostal muscles. The chest cavity is entered through 4th-5thintercostal space, and the incision expanded to allow visualization ofthe heart. The pericardium is opened to expose the heart.

A 6-0 silk suture with a taper needle is passed around the left coronaryartery near its origin, about 1 mm from the insertion of the leftauricular appendage. The coronary artery is occluded by tying the suturearound the artery. The ECG is monitored for S-T changes indicative ofischemia. If the animal develops ventricular fibrillation, gentlecardiac massage is used to convert the animal to a normal rhythm. Shamoperated controls are subjected to the same procedure, but the suture isnot tied off. The incision is closed in three layers. Infected ormoribund animals are eliminated from the study.

Four weeks after surgery, the animals are anesthetized, and a catheteris placed in the right carotid artery and advanced into the leftventricle for hemodynamic measurements. Pressure traces are recorded andanalyzed for heart rate, left ventricular systolic and diastolicpressure, left ventricular developed pressure, and dP/dt max and min.After measurements are taken, 2 ml blood is removed and placed in serumand plasma tubes. The heart is removed and placed on a Langendorffapparatus as follows:

Langendorff Procedure

Buffer preparation Krebs-Henseleit (KH) buffer solution containing NaCl118 mmol/L, KCl 4.7 mmol/L, MgSO4 1.2 mmol/L, KHPO4 1.2 mmol/L, Glucose11 mmol/L, NaHCO3 25 mmol/L and CaCl2 2.5 mmol/L (Sigma) was made upfresh each day using Nanopure pyrogen-free water.

The animal receives 200 units of heparin, the thorax is opened and theheart is rapidly excised and placed in ice-cold KH buffer solution.After the contractile activity of the heart completely ceases, the heartis trimmed and the ascending aorta freed from the connective tissue. Theheart is quickly weighed, then the aorta is cannulated, and the heartmounted on a non-recirculation Langendorff perfusion apparatus (RadnotiGlass Technology, Inc., Monrovia, Calif.). The heart is perfused in aretrograde fashion via the aorta with KH buffer solution oxygenated with95% O₂ and 5% CO₂ to maintain pH 7.4 at 37° C. To assess contractilefunction, a latex balloon is inserted into the left ventricle throughthe mitral orifice and connected to a pressure transducer by rigidpolyethylene tubing. The balloon is inflated with water to a leftventricular end-diastolic pressure (LVEDP) of 1 to 10 mm Hg. Flow isinitiated at 12 ml/min and adjusted during the first 15 minutes ofbaseline to obtain a perfusion pressure between 65 and 75 mmHg. Targetparameters for baseline are as follows:

Perfusion pressure 65-75 mmHg

LVEDP 10 mmHg

The heart is allowed to stabilize for 15 minutes. After this timefunctional measurements are taken, after which a pressure volume curveis generated by adjusting the volume in the balloon in 0.05 mlincrements and recording ventricular pressures. The left ventricularsystolic pressure (LVSP), left ventricular end diastolic pressure(LVEDP), left ventricular developed pressure (LVDP), first derivative ofthe rise and fall in the left ventricular pressure (dp/dt max, dp/dtmin), perfusion pressure and heart rate are automatically recorded usinga computerized data acquisition system.

Other Measurements

After removal, the heart, lungs and liver are weighed. The lungs andliver are weighed and dried overnight for determination of wet to dryratios.

After completing the Langendorff procedure, the heart is placed in coldsaline to stop the beating, then cut into five 2-mm thick transverseslices from apex to base. Slice #3 will be incubated in 1%triphenyltetrazolium chloride (TTC) in 0.9% saline for 20 minutes at 37°C. Tetrazolium reacts with NADH in the presence of dehydrogenase enzymescausing viable tissue to stain a deep red color and that is easilydistinguished from the infarcted pale-unstained necrotic tissue. Theslice is placed apex side down in the lid of a small petri dish for thestaining procedure. The bottom of the dish is placed over the slice tokeep it flat. The slice is then photographed and the areas of infarctedtissue, left and right ventricle are determined using a computerizedimage analysis system. Infarct size is expressed as a percentage of thetotal ventricle. Total area of the left and right ventricle is measured.The remaining sections are divided into right and left ventricle andfrozen for thiobarbituric reactive substances (TBARS) and glutathioneassays.

Treatment Protocol

No treatment is given to the sham operated and control groups.

Measurements for CHF Study

In vivo measurements are made of heart rate (HR), left ventricularsystolic pressure (LVSP), left ventricular end diastolic pressure(LVEDP), dP/dt min and max, right ventricular systolic pressure (RVSP),right ventricular diastolic pressure (RVDP), and right ventricular enddiastolic pressure (RVEDP), as well as total body weight. Ex vivomeasurements are made of HR, LVSP, LVEDP, dP/dt min and max, andpressure volume curve. Also measured ex vivo are heart weight, infarctsize, Glutathione perosidase (GPX), catalase, thiobarbituric reactivesubstances (TBARS), glutathione ratio (GSH/GSSG), lung and liver wet todry weight ratios, serum isoprostane and interleukin-6 (IL-6).

The compounds of the present invention can be tested by this method.

Example 41 Interleukin-1.Beta. Microglial Cell Assay

Materials and Equipment

A. Materials for Cell Preparation and Experiment

-   -   Mouse microgial cell line    -   DMEM High Glucose media (Gibco Catalog #11965-092)    -   FBS (Hyclone Catalog #SH30070.03)    -   100× Penicillin/Streptomycin (Gibco Catalog #15140-122).    -   LPS (Sigma Catalog #L2537)    -   Interferon-gamma (Sigma Catalog #14777)    -   Cell Tracker Green (Molecular Probes Catalog #C2925)    -   HBSS buffer (950 ml Pyrogen-free water, 2.44 g/L MgCl2.6H20,        3.73 g/L KCl, 59.58 g/L Hepes, 58.44 g/L NaCl, 1.36 g/L KH2PO4,        1.91 g/L CaCl2 .2H2O and pH to 4.5 with HCl)    -   Sterile 96-well plates precoated with poly-D-lysine (Corning        Catalog #3665)    -   96-well deep well mother plate, DyNA Block 1000 (VWR Catalog        #40002-008)        B. Materials for Il-1 Beta ELISA    -   Mouse IL-1 beta Duo Set (R & D Systems Catalog #DY401)    -   Substrate Solution (R & D Systems Catalog #DY 999)    -   Bovine Serum Albumin fraction V (BSA V) (Sigma Catalog #A4503)    -   96-well Costar EIA high binding plates (VWR Catalog #29442-302)    -   Plate seal (VWR Catalog #29442-310)    -   PBS (Irvine Scientific Catalog #9240)    -   Cell Culture Grade Water (Irvine Scientific Catalog #9312)    -   Tween 20 (Sigma Catalog #P 1379)    -   Sucrose (Sigma Catalog #S7903)    -   Sodium Azide (Sigma Catalog #S 8032)    -   H₂SO₄ 5N (VWR Catalog #JT 5691-2)        Experimental Preparation and Procedure:

LPS Activation:

Mouse microglial cells were seeded in poly-D-lysine coated 96-wellplates at a density of 10,000 cells/well and allowed to attach for 24hours. Cells were stimulated by addition of LPS (10 μg/ml) and IFN gamma(10 ng/ml) in the presence or absence of test article. The cells werethen incubated for 24 hours at 37° C., after which time the media wasremoved and used for cytokine determination as described below.

Cell Viability:

Viability of mouse microglial cells after exposure to the test articlewas determined using a fluorescent viability dye, Cell Tracker Green.Cell Tracker Green was used at a working concentration of 5 μM in 1×HBSS. Cells were washed once with HBSS (200 μl/well) and 100 μl CellTracker Green was added to each well. Cells were then incubated at 37°C. for 30 minutes, after which time the Cell Tracker was removed and thecells were washed once with HBSS (200 μl/well). 100 μl fresh HBSS wasadded to each well and the plate was read on a Fluoroskan plate readerusing an excitation wavelength of 485 nm and an emission wavelength of538 nm.

Mouse IL-1Beta Elisa:

Solutions:

Wash Buffer: PBS 1L+500 μl Tween 20 (final 0.05%) pH 7.2-7.4.

Blocking Buffer: 500 ml PBS+5 g BSA V (1%)+25 g Sucrose (5%)+0.25 gSodium Azide (0.05%).

Reagent Diluent: 500 ml PBS+5 g BSA V (1%) pH 7.2-7.4 and filtersterilize through 0.2 μm.

Stop Solution: 2N sulfuric acid.

Duo Set Preparations:

1. The IL-1.beta. capture antibody was reconstituted in 1 ml of PBS togive a final concentration of 720 μg/ml, and the working concentrationwas 4 μg/ml. For coating one 96-well plate (at 100 μl/well) 56 μl of the720 μg/ml stock was diluted into 10 ml of PBS.2. The IL-1.beta. standards were reconstituted in 0.5 ml of ReagentDiluent (70 ng/ml). For a high standard of 1 ng/ml (2 wells at 100 μleach+enough for series dilution) 7.1 μl of the 70 ng/ml standard werediluted into 0.5 ml of Reagent Diluent3. The IL-1.beta. detection antibody was reconstituted in 1 ml ofReagent Diluent to give a final concentration of 18 μg/ml and theworking concentration is 100 ng/ml. For one 96-well plate (at 100μl/well) 56 μl of the 18 μg/ml stock was diluted into 10 ml of ReagentDiluent.

IL-1.Beta ELISA Procedure:

Plate Preparation:

1. The Costar EIA Hi-binding plate was coated with capture antibody at 4μg/ml. Each well was coated with 100 μl, and the plate was sealed andincubated overnight at room temperature.

2. Each well was aspirated and washed 3× with Wash Buffer. Each well wasfilled to the top, dispensed, and any remaining buffer was removed byinverting the plate and gently blotting against clean paper towels.

3. Non-specific binding sites were blocked by adding 300 μl of BlockingBuffer to each well, and after sealing incubating for at least 1 hour atroom temperature.

4. After washing the plate was now ready for the samples.

Assay Procedure:

5. 100 μl of either standard or sample were added in each well of thecapture-coated and pre-blocked plate. The plate was sealed and incubatedfor 2 hours at room temperature, followed with washing as in step 2.

6. 100 μl of the detection antibody (100 g/ml) were added to each well.

7. The plate was sealed and incubated at room temperature for 2 hours,followed with washing as in step 2.

8. 100 μl of the working dilution of Streptavidin-HRP was added, and theplate was sealed and incubated in the dark for 20 minutes at roomtemperature, followed with washing as in Step 2.

9. The fresh Substrate Solution was prepared by mixing Color Reagent A(H₂O₂) and Color Reagent B (Tetramethylbenzidine) in a 1:1 ratio. 100 μlof this Substrate Solution mixture was added to each well and the platewas incubated in the dark for 20 minutes at room temperature.10. 50 μl of Stop Solution was added to each well, mixing was ensured bygently tapping.11. Each plate was read with the Spectramax once at 450 nm.Results

When tested as described above, compounds of the present invention, suchas:

-   3-(2-chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(4-fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   4-(benzooxazol-2-ylsulfanyl)-5-(benzooxazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   3-(2-chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2,4-dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(benzooxazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid methyl ester;-   4-hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-dimethyl-propionyloxy)-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-butylsulfanyl-2-butylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-3-(4-methoxy-benzylsulfanyl)-2-(4-methoxy-benzylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-5-oxo-3-(1-oxy-pyridin-2-ylsulfanyl)-2-(1-oxy-pyridin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-hydroxy-3-(4-methyl-pyrimidin-2-ylsulfanyl)-2-(4-methyl-pyrimidin-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester,-   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid isopropyl ester; and-   4-Hydroxy-3-(1-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester    inhibited the IL-1beta induction with an EC₅₀ of 20 μM or less.

Example 42 Rat Paw Edema Assay

Animal Preparation:

Male Sprague-Dawley rats weighing between 175 to 200 g are used in thisstudy. Animals are allowed free access to water and commercial rodentdiet under standard laboratory conditions. Room temperature ismaintained at 20-23° C. and room illumination is on a 12/12-hourlight/dark cycle. Animals are acclimatized to the laboratory environment5 to 7 days prior to the study.

Experimental Procedure:

Each animal is treated by administration of vehicle, reference or testsubstance one hour prior to carrageenan injection, as follows:

I.V. Infusion via Femoral Vein: Anesthesia is maintained by inhalationof 3.0% isoflurane (Aerrane, Front Dodge, Iowa) in oxygen throughout theentire procedure. The exterior site of the right femoral vein is shavedand sterilized prior to surgery. A 3-cm incision is made in the rightgroin region and the femoral vein is isolated. The femoral vein istemporarily ligated with a micro-vascular clip, and a small incision ismade on the femoral vein to introduce and advance a polyethylene (PE-50)catheter (Becton. Dickinson and Co., Sparks, Md.). The catheter issecured in place with suture (silk 5/0, Carlisle Laboratories, FarmersBranch, Tex.). The other end of the catheter is attached to a syringefilled with the saline for the bolus injection. Using a hemostat, apocket is made subcutaneously on the back of the animal so the PEcatheter can be brought up to the exteriorization point between theshoulder blade for either a bolus injection or a continuous injection byan osmotic pump.

I.P. Injection: An awake rat is held in a standard hand held position. A23¾G needle is injected into the lower right quarter of the abdomen passthe peritoneum, slightly off the midline. To avoid organ injection, theplunger of the syringe is slightly pulled back. If no fluid iswithdrawn, the content of the syringe is delivered into the abdominalcavity.

Gavage Feeding: A standard rat gavage tube (Popper & Sons Inc, NY) isattached to a 3-cc hypodermic syringe. The animal is held in a verticalposition. The feeding tube is placed into the mouth and then gentlyadvanced until it reaches the stomach (the approximate insertion lengthof the tube should be measured prior to feeding). The content of thesyringe is slowly delivered, and then the tube is withdrawn.

One hour post treatment each animal is anesthetized with 3.0% isoflurane(Aerrane, Front Dodge, Iowa) in oxygen and administered 100 μl of 1%Carrageenan Lambda type IV (Sigma Chemical Company, St. Louis, Mo.)suspension in saline, into the intraplantar surface of the right hindpaw. Paw edema is measured four hours after carrageenan injection,either by measuring the increase in paw volume using a plethysmometer orthe increase in paw weight using a fine scale. Immediately prior toedema measurement, the animals are euthanized via CO₂ asphyxiation and500 μl blood is withdrawn by cardiac puncture for later analysis. Pawvolume is determined by the extent to which water is displaced by thepaw from a pre-calibrated chamber. The volume of the left hind paw(control) is subtracted from the volume of the right hind paw(carrageenan-treated) to determine the volume of carrageenan-inducededema. To measure the weight difference between paws, both hind pawswere removed and weighed separately.

To minimize the variation in the model following steps are taken:

-   -   Carrageenan is made fresh every day prior to the study (2-3        hours before injection).    -   The plethysmometer is calibrated each day prior to the study.    -   If carrageenan injection causes significant bleeding or a        hematoma on the treated foot, the animal is excluded from the        study.    -   Each paw is marked at the tibio-tarsal joint across the ankle        prior to measurements, to ensure each paw is submerged at the        same level.    -   If reading on the volume needs to be repeated, the paw must be        dried off completely.        Statistical Analysis

The difference of the weight or the volume between right and left paw iscalculated for each animal for the analysis. Group data are presented asmeans+/−SEM and p<0.05 are considered significant. Inter-groupcomparisons are carried out by unpaired student t test (between twogroups) or one-way ANOVA followed by post hoc Bonferroni's multiplecomparisons.

Results

Certain compounds of the present invention showed activity when testedby this method.

Example 43 Mouse Ear Inflammatory Response to Topical Arachidonic Acid

Animals: Balb C Mice 23-28 gms, from Simonsen Labs, Gilroy, Calif.

Materials:

Arachidonic Acid, 99% pure from Porcine Liver (Sigma Aldrich)reconstituted in acetone 2 mg/20 ul (200 mg/ml).

Inhalation anesthesia: Isoflurane 3% (Baxter).

Blood Sample tubes: Microtainer tubes w/ heparin (Becton Dickinson).

TNFa Elisa assay (R&D Science).

Experimental Procedure

Test compounds, positive control (arachidonic acid only) and standard(Dexamethasone @0.1 mg/kg) prepared in solutions of acetone, ethanol oraqueous ethanol, are applied to both sides of the right ear with anEppendorf repipettor pipette, in a volume of 10 μl each side (20 μltotal). 30 Minutes later, 10 μl of arachidonic acid is applied to bothsides of the right ear (20 μl total). One hour after the application ofarachidonic acid, the mice are deeply anesthetized with isoflurane and ablood sample is taken via the orbital sinuses and placed in Microtainertubes. The animals are then euthanized by CO₂ inhalation and the rightears removed at the base. A uniform plug of ear tissue is obtained usinga 8 mm dermal punch. The earplugs are quickly weighed to the nearest 0.1mg and then flash frozen for TNFα determination.

Statistical Analysis:

Group data are presented as mean+/−SEM and p<0.05 is consideredsignificant. Inter-group comparisons are carried out by unpaired studentt tests (between two groups) or ANOVA (three or more groups) followed bypost hoc Dunnet's test.

Results

The compounds of the present invention, such as:

-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanyl)-2-(5-phenyl-2H-[1,2,4]triazol-3-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-5-(1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrimidin-2-ylsulfanyl)-2-(pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(5-sulfonic    acid-1H-benzoimidazol-2-ylsulfanyl)-2-(5-sulfonic    acid-1H-benzoimidazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester, hydrochloride salt;-   4-Hydroxy-5-oxo-3-(pyridin-4-ylsulfanyl)-2-(pyridin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   5,8-Dichloro-3-hydroxy-2-oxo-2H-1-oxa-4,9-dithia-benzo[f]azulene-10a-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Amino-[1,3,4]thiadiazol-2-ylsulfanyl)-2-(5-amino-[1,3,4]thiadiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;    showed significant reduction in edema (10 to 70%, p<0.05) when    tested by this method.

Example 44 Skin Protection Assay

Cytoprotective activity for skin can be evaluated in cell culture usingthe Epiderm Skin Model (EPI-100) from the Mattek Corporation of Ashland,Mass. Cell cultures of neonatal foreskin are cultured in accordance withthe manufacturer's directions, and are assayed for percent cellularviability by measuring the amount of3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dyetaken up by the cells. Viable cells take up this dye and convert it toinsoluble formazin crystals that reside in the mitochondria of the cellsuntil extracted with alcohol. The amount of MTT converted to extractableformazin crystals is directly proportional to the viability of the cellculture. MTT is measured spectrophotometrically.

Cells are exposed to UV light at a rate of 1.5 Minimal Erythemal Dose(MED) per hour per square centimeter, for a total dose of about 31.5mJ/cm², from a solar simulator (filtered to yield wavelengths in theregion of 290-400 nm) in the presence of the cytoprotective compound ormixtures thereof to measure the effect of test compounds to protect thecell culture from the generation of free radicals resulting from theultraviolet light.

The controls for this study are cell cultures without added testcompound (positive control). All cell cultures are also compared tocultures that are not exposed to UV light and do not include thecytoprotective agents or blends in order to determine percent cellularviability (negative control). This latter measurement is assumed to beequal to 100% viability.

Cell cultures treated with certain furanone compounds of the inventionshow greater survival than do positive control cell cultures, whentested as described above.

Example 45 E-Selectin Cell Inflammation Assay

Endothelial-Leukocyte Adhesion Molecule (ELAM), also known asE-selectin, is expressed on the surface of endothelial cells. In thisassay, lipopolysaccharide (LPS) and IL-1β are used to stimulate theexpression of ELAM; test agents are tested for their abilities to reducethis expression, in accordance with studies showing that reduction ofleukocyte adhesion to endothelial cell surface is associated withdecreased cellular damage (e.g., Takada, M., Et al., Transplantation 64:1520-25, 1997; Steinberg, J. B., et al., J. Heart Lung Trans.13:306-313, 1994).

Endothelial cells may be selected from any of a number of sources andcultured according to methods known in the art; including, for example,coronary artery endothelial cells, human brain microvascular endothelialcells (HBMEC; Hess, D.C., et al., Neurosci. Left. 213(1): 37-40, 1996),or lung endothelial cells. Cells are conveniently cultured in 96-wellplates. Cells are stimulated by adding a solution to each wellcontaining 10 μg/ml LPS and 100 pg/ml IL-1β for 6 hours in the presenceof test agent (specific concentrations and time may be adjusteddepending on the cell type). Treatment buffer is removed and replacedwith pre-warmed Fixing Solution® (100 μl/well) for 25 minutes at roomtemperature. Cells are then washed 3×, then incubated with BlockingBuffer (PBS+2% FBS) for 25 minutes at room temperature. Blocking Buffercontaining Monoclonal E-Selectin Antibody (1:750, Sigma Catalog #S-9555)is added to each well. Plates are sealed and stored at 4° overnight.Plates are washed 4× with 160 μL Blocking Buffer per well. SecondAntibody-HRP diluted 1:5000 in Blocking Buffer is then added (100μL/well), and plates are incubated at room temperature (protected fromlight) for two hours. Plates are then washed 4× with Blocking Bufferbefore addition of 100 μL of ABTS Substrate solution at room temperature(Zymed, Catalog #00-2024). Wells are allowed to develop for 35 minutes,before measurement at 402 nm in a Fluoroskan® Reader with shake programfor 10 seconds. Positive results are recorded as a decrease in ELAMconcentration in tested wells, as compared to control wells.

In an ELAM assay, such as the one described herein, the followingcompounds of the present invention were able to reduce the expression ofELAM at EC₅₀ of 30 μM or less:

-   R-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)    ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-oxy-pyridin-2-ylsulfanyl)-2-(1-oxy-pyridin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-5-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;-   3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(Benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   Dimethylamino-acetic acid    3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethyl    ester;-   3-(2-Chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid;-   3-(5,6-Dichloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5,6-dichloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-Benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid 2-isopropyl-5-methyl-cyclohexyl ester;-   Di-[2-(4-Hydroxy-5-oxo-2-carboxylic acid methyl ester)]-disulfide;-   3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylic    acid diethyl ester;-   4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4,5]dec-3-ene-2,6,8,10-tetraone;-   3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic    acid isopropyl ester;-   4-Acetoxy-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-ethyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester;-   4-Acetoxy-3-benzylsulfanyl-2-benzylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester; and-   4-Hydroxy-3-(1-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylic    acid ethyl ester.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto. All patents and publications cited above arehereby incorporated by reference.

1. A method of treatment for a mammal suffering from a dermatologiccondition selected from the group consisting of contact dermatitis,acne, rosacea, and psoriasis, comprising administering a therapeuticallyeffective amount of a compound of Formula I:

wherein: R¹ is: C(O)OR′; —C(O)NR′R″; —CH₂OR′″; cyano; optionallysubstituted heterocyclyl; optionally substituted heterocyclyl-alkyl;optionally substituted heteroaryl, or optionally substitutedheteroaralkyl; R² is: optionally substituted alkyl; optionallysubstituted cycloalkyl; optionally substituted aryl; optionallysubstituted aralkyl; optionally substituted heterocyclyl; optionallysubstituted heteroaryl; optionally substituted heteroaralkyl; anoptionally substituted nucleoside; an optionally substituted amino acid;or an optionally substituted di-, tri- or tetra-peptide; R³ is:optionally substituted alkyl; optionally substituted cycloalkyl;optionally substituted aryl; optionally substituted aralkyl; optionallysubstituted heterocyclyl; optionally substituted heteroaryl; optionallysubstituted heteroaralkyl; an optionally substituted nucleoside; anoptionally substituted amino acid; or an optionally substituted di-,tri- or tetra-peptide; R⁴ is: hydrogen; alkyl; alkylcarbonyl;(poly)alkoxyalkylene; or dialkoxyphosphoryloxy; X is: —S—; —S(O)—;—S(O)₂—; or X taken together with R² is —P(O)(OR′)₂; Y is: —S—; —S(O)—;—S(O)₂—; or Y taken together with R³ is —P(O)(OR′)₂; or X—R² takentogether with Y—R³ form an optionally substituted aliphatic or aromaticring; R′ is: hydrogen; alkenyl; optionally substituted alkyl; optionallysubstituted cycloalkyl; phosphoryl; or optionally substituted aryl; R″is: hydrogen; alkenyl; optionally substituted alkyl; or optionallysubstituted aryl; or R′ and R″ together with the atom to which they areattached form a 5- to 7-membered aromatic, saturated or unsaturatedring, optionally incorporating one or more additional heteroatoms chosenfrom N, O, or S, and optionally substituted with one or moresubstitutents selected from the group consisting of optionallysubstituted lower alkyl, halo, cyano, alkylthio, lower alkoxy, carboxy,benzyl, and oxo; R′″ is: hydrogen; alkenyl; optionally substitutedalkyl; acyl, optionally substituted cycloalkyl; phosphoryl; oroptionally substituted aryl; or a compound of Formula III:

wherein: R⁵ is: —C(O)OR^(a); —C(O)NR^(a)R^(b); CH₂OR^(d); —C(O)R^(c);cyano; optionally substituted heterocyclyl; or optionally substitutedheteroaryl; R⁶ is: hydrogen; —C(O)OR^(a); —C(O)NR^(a)R^(b); CH₂OR^(d);—C(O)R^(c); cyano; optionally substituted alkyl; optionally substitutedheterocyclyl; optionally substituted aryl, or optionally substitutedheteroaryl; or R⁵ and R⁶ with the atom to which they are attached forman optionally substituted ring; R⁷ is: optionally substituted alkyl;optionally substituted cycloalkyl; optionally substituted aryl;optionally substituted aralkyl; optionally substituted heterocyclyl;optionally substituted heteroaryl; optionally substituted heteroaralkyl;an optionally substituted nucleoside; an optionally substituted aminoacid; or an optionally substituted di-, tri- or tetra-peptide; with theproviso that when R⁶ is alkyl, then R⁷ is optionally substitutedheterocyclyl, optionally substituted heteroaryl, or optionallysubstituted heteroaralkyl, or R⁵ and R⁷ with the atoms to which they areattached form an optionally substituted heterocyclic ring; R⁸ is:hydrogen; alkyl; alkylcarbonyl; (poly)alkoxyalkylene; ordialkoxyphosphoryloxy; Y′ is: S—; R^(a) is: hydrogen; alkenyl;optionally substituted alkyl; optionally substituted cycloalkyl; oroptionally substituted aryl; R^(b) is: hydrogen; alkenyl; optionallysubstituted alkyl; or optionally substituted aryl; or R^(a) and R^(b)together with the atom to which they are attached for a 5- to 7-memberedaromatic, saturated or unsaturated ring, optionally incorporating one ormore additional heteroatom chosen from N, O, or S, and optionallysubstituted with one or more substituents selected from the groupconsisting of optionally substituted lower alkyl, halo, cyano,alkylthio, lower alkoxy, carboxy, benzyl, and oxo; R^(c) is: optionallysubstituted alkyl or optionally substituted aryl; and R^(d) is:hydrogen; alkenyl; optionally substituted alkyl; acyl; optionallysubstituted cycloalkyl; or optionally substituted aryl; including singletautomers; single stereoisomers and mixtures of tautomers and/orstereoisomers, and the pharmaceutically acceptable salts thereof.
 2. Themethod of claim 1, wherein X and Y are both —S—.
 3. A method oftreatment for a mammal suffering from a dermatologic condition selectedfrom the group consisting of contact dermatitis, acne, rosacea, andpsoriasis comprising administering a therapeutically effective amount ofa compound selected from the group consisting of:R-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl-)ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylic acidethyl ester;3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(1-oxy-pyridin-2-ylsulfanyl)-2-(1-oxy-pyridin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-5-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;3-(Furan-2-ylmethysulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;2-(Benzothiazole-2-sulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanymethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester; Dimethylamino-acetic acid3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethylester;3-(2-Chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid;2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid;3-(5,6-Dichloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5,6-dichloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4,5]dec-3-ene-2,6,8,10-tetraone;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid isopropyl ester;4-Acetoxy-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Acetoxy-3-benzylsulfanyl-2-benzylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(1-methyl-1H-benzoimida-zol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(1H-Benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid 2-isopropyl-5-methyl-cyclohexyl ester;3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylicacid diethyl ester.
 4. A method of treatment for a mammal suffering froma dermatologic condition selected from the group consisting of contactdermatitis, acne, rosacea, and psoriasis comprising administering atherapeutically effective amount of a compound selected from the groupconsisting of:3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5,-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-fur-an-2-carboxylicacid ethyl ester;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Chloro-phenysulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-pheny-1-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid isopropyl ester;4-Hydroxy-3-(1-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanymethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid 2-isopropyl-5-methyl-cyclohexyl ester;3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2,-dicarboxylicacid diethyl ester.
 5. The method of claim 1, comprising topicallyadministering at least one compound of Formula I or of Formula III. 6.The method of claim 3, comprising applying to the area of skin in needof such treatment a composition comprising at least one compoundselected from the group consisting of:R-3-[2-(4-Amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanyl]-2-[2-(4-amino-4-carboxy-butyrylamino)-2-(carboxymethyl-carbamoyl)-ethylsulfanylmethyl]-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(5-Chloro-benzothiazol-2-ylsulfanyl)-2-(5-chloro-benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Cyclohexylsulfanyl-2-cyclohexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(pyrrolidine-1-carbothioylsulfanyl)-2-(pyrrolidine-1-carbothioylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzoselenazol-2-ylsulfanyl)-2-(benzoselenazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(7-trifluoromethyl-quinolin-4-ylsulfanyl)-2-(7-trifluoromethyl-quinolin-4-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(2-sulfo-ethylsulfanyl)-2-(2-sulfo-ethylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(4-trifluoromethyl-pyrimidin-2-ylsulfanyl)-2-(4-trifluoromethyl-pyrimidin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(1-oxy-pyridin-2-ylsulfanyl)-2-(1-oxy-pyridin-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Chloro-phenylsulfanyl)-2-(2-chloro-phenylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Hexylsulfanyl-2-hexylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(1-phenyl-1H-tetrazol-5t-ylsulfanyl)-2-(1-phenyl-1H-tetrazol-5-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(naphthalen-2-ylsulfanyl)-2-(naphthalen-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-5-oxo-3-(4-phenyl-thiazol-2-ylsulfanyl)-2-(4-phenyl-thiazol-2-ylsulfanylmethyl)-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(5-sulfo-1H-benzoimidazol-2-ylsulfanyl)-5-(5-sulfo-1H-benzoimidazol-2-ylsulfanylmethyl)-3-hydroxy-5-hydroxymethyl-5H-furan-2-one;3-(Furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-4-isobutyryloxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanyl]-2-[4-(2-methoxycarbonyl-vinyl)-phenylsulfanylmethyl]-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(6-nitro-benzothiazol-2-ylsulfanyl)-2-(6-nitro-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;2-(Benzothiazole-2-fulfinylmethyl)-3-(benzothiazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2,4-Dichloro-benzylsulfanyl)-2-(2,4,-dichloro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(5-methoxy-benzothiazol-2-ylsulfanyl)-2-(5-methoxy-benzothiazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Chloro-6-fluoro-benzylsulfanyl)-2-(2-chloro-6-fluoro-benzylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester; Dimethylamino-acetic acid3-(1H-benzoimidazol-2-ylsulfanyl)-2-(1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-ylmethylester;3-(2-Chloro-4-fluoro-phenylsulfanyl)-2-(2-chloro-4-fluoro-phenyslsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid;2-(Furan-2-ylmethanesulfinylmethyl)-3-(furan-2-ylmethanesulfonyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(furan-2-ylmethanesulfinyl)-2-(furan-2-ylmethanesulfinylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(2,2-dimethyl-propionyloxy)-3-ethoxycarbonylmethylsulfanyl-2-ethoxycarbonylmethylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(2-Dimethylamino-ethylsulfanyl)-2-(2-dimethylamino-ethylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid;3-(5,6-Dichloro-1H-benzoimidazol-2-ylsulfanyl)-2-(5,6-dichloro-1H-benzoimidazol-2-ylsulfanylmethyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-Benzylsulfanyl-2-benzylsulfanylmethyl-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-(1H-Benzoimidazol-2-ylsulfanyl)-3-hydroxy-1-oxa-7,9-diaza-spiro[4,5]dec-3-ene-2,6,8,10-tetraone;3-(Benzothiazol-2-ylsulfanyl)-2-(benzothiazol-2-ylsulfanylmethyl)-4-hydro-xy-5-oxo-2,5-dihydro-furan-2-carboxylicacid isopropyl ester;4-Acetoxy-3-(furan-2-ylmethylsulfanyl)-2-(furan-2-ylmethylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Ethoxy-3-(1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-ethyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;2-(1H-Benzoimidazol-2-ylsulfanylmethyl)-4-ethoxy-3-1-ethyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Acetoxy-3-benzylsulfanyl-2-benzylsulfanylmethyl-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(1-methyl-1H-benzoimidazol-2-ylsulfanyl)-2-(1-methyl-1H-benzoimidazol-2-ylsulfanylmethyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;3-(1H-Benzoimidazol-2-ylsulfanyl)-4-hydroxy-5-oxo-2,5-dihydro-furan-2-carboxylicacid ethyl ester;4-Hydroxy-3-(5-methyl-1H-benzoimidazol-2-ylsulfanyl)-5-oxo-2,5-dihydro-furan-2-carboxylicacid 2-isopropyl-5-methyl-cyclohexyl ester;3-(4-Fluoro-benzylsulfanyl)-4-hydroxy-5-oxo-5H-furan-2,2-dicarboxylicacid diethyl ester.
 7. The method of claim 1, comprising administering aformulation for topical application comprising one or more topicalexcipients and at least one compound of Formula I or of Formula III. 8.The method of claim 1, additionally comprising an additional benefitagent, selected from the group consisting of sunscreens, retinoid andderivatives thereof, antioxidants, hydroxyacids, botanical extracts,salicylic acid, benzoyl peroxide, antibiotics, antiandrogens,anti-inflammatory agents, vitamins, tocopherol (α-, β-, γ-, δ-) andesters thereof, corticosteroids and mixtures thereof.
 9. The method ofclaim 3, additionally comprising an additional benefit agent, selectedfrom the group consisting of sunscreens, retinoid and derivativesthereof, antioxidants, hydroxyacids, botanical extracts, salicylic acid,benzoyl peroxide, antibiotics, antiandrogens, anti-inflammatory agents,vitamins, tocopherol (α-, β-, γ-, δ-) and esters thereof,corticosteroids and mixtures thereof.
 10. The method of claim 3,comprising administering a formulation for topical applicationcomprising one or more topical excipients and at least one compound ofFormula I or of Formula III.
 11. The method of claim 4, additionallycomprising an additional benefit agent, selected from the groupconsisting of sunscreens, retinoid and derivatives thereof,antioxidants, hydroxyacids, botanical extracts, salicylic acid, benzoylperoxide, antibiotics, antiandrogens, anti-inflammatory agents,vitamins, tocopherol (α-, β-, γ-, δ-) and esters thereof,corticosteroids and mixtures thereof.
 12. The method of claim 4,comprising administering a formulation for topical applicationcomprising one or more topical excipients and at least one compound ofFormula I or of Formula III.